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Running title: 1
FTSH4 as a mitochondrial quality control protein 2
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Corresponding authors: 5
Hanna Janska 6
Faculty of Biotechnology, University of Wroclaw 7
F. Joliot-Curie 14A, 50-383 Wroclaw, Poland 8
Tel. + 48 71 375 62 49, Fax + 48 71 375 62 34 9
E-mail address: [email protected] 10
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Christiane Funk 12
Dept. of Chemistry, Umeå University 13
901 87 Umeå, Sweden 14
Tel. + 46 90 786 7633, Fax + 46 90 786 7655 15
E-mail address: [email protected] 16
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Plant Physiology Preview. Published on June 13, 2016, as DOI:10.1104/pp.16.00370
Copyright 2016 by the American Society of Plant Biologists
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Title: Lack of FTSH4 protease affects protein carbonylation, mitochondrial 27
morphology and phospholipid content in mitochondria of Arabidopsis: new insights 28
into a complex interplay 29
Authors: Elwira Smakowska1*, Renata Skibior-Blaszczyk1*, Malgorzata Czarna1, 30
Marta Kolodziejczak1, Malgorzata Kwasniak-Owczarek1, Katarzyna Parys1, 31
Christiane Funk2a, Hanna Janska1a 32
1Faculty of Biotechnology, University of Wroclaw, F. Joliot-Curie 14A, 50-383 33
Wroclaw, Poland 34
2Department of Chemistry, Umeå University, 901 87 Umeå, Sweden. 35
aCorresponding authors 36
* These authors contributed equally to this work 37
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Summary Sentence: FTSH4 protease controls a proper cardiolipin content in the 39
mitochondrial membrane and in consequence prevents oxidative stress. 40
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Author contributions 42
HJ, CF designed the research. ES, RS, MC, MK, MKO, KP performed the 43
experiments. HJ, ES, MC, RS, MK, MKO, KP analyzed the data. HJ wrote the article 44
with input of all co-authors. 45
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This work was supported by Grants 2011/03/N/NZ2/00221 and 47
2013/11/N/NZ3/00061 from the National Science Centre, Poland, as well as by the 48
Swedish Energy Agency (2012-005889) and Umeå University. The contribution of 49
MKO was supported by the START scholarship granted by Foundation for Polish 50
Science and scholarship for outstanding young scientists from the Polish Minister of 51
Science and Higher Education. 52
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The Proteomics Facility of the Chemical Biological Centre of Umeå University 54
provided the facilities for performing the 2D-DIGE experiment and the MALDI-TOF 55
mass spectrometry analyses. 56
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Corresponding authors: 58
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ABSTRACT 92
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FTSH4 is one of the inner membrane-embedded ATP-dependent 94
metalloproteases in mitochondria of Arabidopsis thaliana. In mutants impaired to 95
express FTSH4 carbonylated proteins accumulated and leaf morphology was altered 96
when grown under short-day photoperiod, at 22°C, and long-day photoperiod, at 97
30°C. To provide better insight into the function of FTSH4 we compared the 98
mitochondrial proteomes and oxyproteomes of two ftsh4 mutants and wild type plants 99
grown under conditions inducing the phenotypic alterations. Numerous proteins from 100
various submitochondrial compartments were observed to be carbonylated in the 101
ftsh4 mutants indicating a widespread oxidative stress. One of the reasons for the 102
accumulation of carbonylated proteins in ftsh4 was limited ATP-dependent proteolytic 103
capacity of ftsh4 mitochondria, arising from insufficient ATP amount, probably as a 104
result of an impaired oxidative phosphorylation (OXPHOS), especially complex V. In 105
ftsh4 we further observed giant, spherical mitochondria co-existing among normal 106
ones. Both effects, the increased number of abnormal mitochondria as well as the 107
decreased stability/activity of the OXPHOS complexes was probably caused by the 108
lower amount of the mitochondrial membrane phospholipid cardiolipin. We postulate 109
that the reduced cardiolipin content in ftsh4 mitochondria leads to perturbations 110
within the OXPHOS complexes generating more reactive oxygen species, less ATP 111
and to deregulation of mitochondrial dynamics causing in consequence the 112
accumulation of oxidative damage. 113
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INTRODUCTION 126
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Mitochondria play an important role in cellular metabolism and cell longevity 128
with the most notable function in the generation of ATP through oxidative 129
phosphorylation. During oxidative phosphorylation (OXPHOS) under unfavorable 130
conditions, reactive oxygen species (ROS) are generated, which might lead to 131
oxidative damages (Moller, 2001). To avoid accumulation of non-functional proteins, 132
particularly the formation of insoluble, harmful protein aggregates, mitochondria have 133
evolved a hierarchically structured quality control (QC) system (Tatsuta and Langer, 134
2008; Baker et al., 2011; Fischer et al., 2012). The QC system links molecular, 135
organellar and cellular levels, and includes: i) a protease/chaperone system (Voos, 136
2013), ii) mitochondrial fission and fusion processes (Twig et al., 2008; Osellame et 137
al., 2012; Youle and van der Blick, 2012, Elgass et al., 2013), and - in case the first 138
two actions fail, iii) initiation of the intrinsic cell death program (Fischer et al., 2012, 139
Gaspard and McMaster, 2015). It ensures the persistence of a healthy mitochondrial 140
population and ultimately survival of the organism, especially during stress conditions 141
(Baker et al., 2014). 142
A conserved intra-mitochondrial network of chaperones and proteases that 143
maintain protein homeostasis (Tatsuta and Langer, 2007; Baker et al., 2011) 144
constitutes the first level of the QC system. Key proteolytic enzymes are ATP-145
dependent proteases, which combine both proteolytic and chaperone-like activities 146
(Voos, 2013). Typically, mitochondria contain three types of ATP-dependent 147
proteases: Lon, Clp, and FtsH (Janska et al., 2010). The two first protease families 148
are classified as serine proteases, whereas FtsH proteases have a catalytic site 149
characteristic for metalloproteases. 150
FtsH proteases, also termed as AAA proteases (ATPases Associated with 151
diverse cellular Activities), form oligomeric complexes in the mitochondrial inner 152
membrane with catalytic domains facing the intermembrane space (i-AAA) or matrix 153
side (m-AAA). Only one i-AAA protease is present in yeast (Yme1) and human 154
(YMEL1), while plant mitochondria contain two i-AAA proteases: FTSH4 and FTSH11 155
(Urantowka et al., 2005). Interestingly, different from human YMEL1, Arabidopsis 156
FTSH4 protease does not complement the yeast homolog, suggesting its plant 157
specific function (Urantowka et al., 2005). Yeast i-AAA protease was reported to act 158
as quality control enzyme and to degrade unassembled inner membrane proteins 159
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(Pearce and Sherman, 1995; Nakai et al., 1995; Kambacheld et al., 2005; Augustin 160
et al., 2005) as well as misfolded small TIM chaperones (translocase of the inner 161
membrane) of the intermembrane space (IMS) (Baker et al., 2012). Non-assembled 162
respiratory chain proteins have been identified as proteolytic substrates for the 163
human i-AAA protease (Stiburek et al., 2012). So far, there is no information about 164
oxidatively damaged proteins being substrates of these i-AAA proteases. However, 165
an increasing amount of evidence indicates their crucial role in mitochondrial quality 166
control (Baker et al., 2011). It seems that the i-AAA protease-mediated proteolysis 167
controls mitochondrial dynamics in multiple ways. Yeast i-AAA was found to degrade 168
the phospholipid transfer proteins UPS1 and UPS2, responsible for the distribution of 169
cardiolipin (CL) and phosphatidylethanolamine (PE) in mitochondrial membranes 170
(Potting et al., 2010). Both phospholipids have a critical impact on several 171
mitochondrial functions including fusion and fission (Joshi et al., 2012; Pan et al., 172
2014). Next, it was shown that Yme1 processes Atg32, a mitochondrial outer 173
membrane receptor ensuring specificity of mitophagy (Wang et al., 2013). 174
Furthermore, in mammals and yeast, i-AAA proteases regulate mitochondrial 175
morphology and dynamics by processing the conserved fusion mediator OPA1 176
(Mgm1 in yeast) (Anand et al., 2013; Ruan et al., 2013). Finally, recent findings 177
indicate that Yme1L regulates mitochondrial dynamics, mitochondrial cristae 178
structure and nucleoid organization by controlling the Mic60/Mitofilin homeostasis (Li 179
et al., 2015). Other postulated biological roles for i-AAA proteases are dependent 180
exclusively on the chaperone-like activity of these enzymes. Specifically, the yeast 181
Yme1 protease has been proposed to act as a chaperone in the folding of numerous 182
proteins in the IMS (Fiumera et al., 2009, Schreiner et al., 2012), and in the assembly 183
of complex V (Francis and Thorsness, 2011). Further, Yme1 was shown to mediate 184
protein import into the intermembrane space (Rainey et al., 2006). 185
In Arabidopsis the loss of FTSH4 protease, one of the two i-AAA proteases in 186
plant mitochondria, impairs development and leaf morphology at the late stage of 187
rosette growth under short-day photoperiod, but not under long days (Gibala et al., 188
2009; Kicia et al., 2010). These conditional, morphological and developmental 189
alternations correlated with elevated levels of ROS and carbonylated proteins, and 190
were accompanied by ultrastructural changes in mitochondria and chloroplasts 191
(Gibala et al., 2009; Kicia et al., 2010). We also found that FTSH4 is significant for 192
assembly/stability of complex I and especially complex V (Kolodziejczak et al., 2007). 193
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More recently, Zhang et al. (2014a) confirmed our observations linking the lack of 194
FTSH4 protease, oxidative stress and alternations in plant development and 195
architecture. These authors also revealed additional non-mitochondrial players such 196
as cytoplasmic peroxidases and auxin homeostasis (Zhang et al., 2014a; Zhang et 197
al., 2014b). They proposed that FTSH4 mediates the peroxidase-dependent interplay 198
between hydrogen peroxide and auxin homeostasis to regulate plant growth and 199
development. 200
In the present study, we compared the mitochondrial proteome and 201
oxyproteome of wild type and ftsh4 mutants grown under conditions inducing the 202
phenotypic alterations in ftsh4 (short days at optimal temperature and long days 203
under continuous moderate heat stress) and found that diverse proteins from various 204
submitochondrial compartments were carbonylated in ftsh4 mitochondria, indicating a 205
widespread oxidative stress. We postulate that this oxidative stress proceeds 206
progressively and is mainly associated with the FTSH4 function to maintain a proper 207
phospholipid content, especially cardiolipin, in the mitochondrial membrane. 208
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RESULTS 228
229
ftsh4 Mutants Show Similar Morphological Changes Under Short-Day 230
Photoperiod at Normal Temperature and in Long Days Under Continuous 231
Moderate Heat Stress 232
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Aging ftsh4-1 and ftsh4-2 Arabidopsis plants grown in short days at normal 234
temperature (SD, 22°C) were previously shown to display severe morphological and 235
developmental alterations compared to wild type (Gibala et al., 2009; Kicia et al., 236
2010). In these studies we further demonstrated that the post-germination growth of 237
ftsh4 under long-day photoperiod (LD, 22°C) was not affected. Here, however, we 238
present data indicating that phenotypic abnormalities are noticeable even in LD, 239
when ftsh4 plants were grown at slightly higher temperature than optimal (30°C). 240
Phenotypic features seen under these both regimens are: a significantly reduced 241
rosette size (Gibala et al., 2009; Fig. 1A and 1C), a developmental delay in the 242
appearance of true leaves (Gibala et al., 2009; Fig. 1B) and irregular serration of leaf 243
blades (Gibala et al., 2009, Fig. 1D), the latter two more visible at the end of 244
vegetative growth. These comparable defects suggest similar molecular alterations 245
activated under two specific conditions in the ftsh4 mutant. We denote these 246
conditions as inducing the phenotype. 247
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Mitochondrial Proteome is Altered in ftsh4 Mutants Under Conditions Inducing 249
the Phenotype 250
251
To gain insight into molecular alterations activated in ftsh4 mutants in 252
conditions inducing the phenotype we analysed their mitochondrial proteomes using 253
Two-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE). 254
Mitochondrial proteins of 9-week-old ftsh4-1 and wild type plants grown in soil under 255
SD, 22°C as well as 3-week-old ftsh4-1, ftsh4-2 and wild type seedlings grown 256
hydroponically under LD, 30°C were differentially labelled using G-Dyes (NH 257
DyeAGNOSTICS) and subjected to IEF/SDS-PAGE. Thus, three experimental setups 258
were analysed (SD, 22°C for ftsh4-1; LD, 30°C for ftsh4-1 and LD, 30°C for ftsh4-2) 259
and protein spots with fold changes in abundance of greater than ±1.2 (p ≤ 0.05) 260
between ftsh4 and wild type in at least one experimental setup were picked from the 261
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gel and identified by MALDI-TOF (PMF, Peptide Mass Fingerprinting). Overall, 63 262
unique and 3 redundant proteins (serine transhydroxymethyltransferase 1, SHM1 - 263
protein spot numbers 28, 29 and 30; ATP synthase subunit 2, ATP2 - protein spot 264
numbers 6, 7, 8, and 9; heat shock protein 60, HSP60-2 - protein spot numbers 38 265
and 39) with significant Mowse scores were identified and classified into different 266
functional categories (Table I, Supplemental Table S1, and Supplemental Fig. S1). 267
Most of these proteins were common to two tested setups and six were common to 268
all three variants assayed. Four of these common proteins are OXPHOS subunits 269
and two are associated with the TCA cycle. These proteins are indicated in a 270
representative analytical 2D-DIGE gel shown in Supplemental Fig. S2. 271
Collectively, several subunits of complex I and V and the majority of enzymes 272
of the TCA cycle were lower in abundance in mitochondria of FTSH4-deficient plants, 273
while chaperones, antioxidant enzymes and proteins involved in the transport 274
accumulated (Table I). A decrease in abundance of the components of OXPHOS and 275
TCA cycle and induction of expression of proteins that have an antioxidant and stress 276
response function is characteristic for mitochondrial proteomes under sub-lethal 277
doses of oxidative stress (Sweetlove et al., 2002). Thus, the proteomic data indicate 278
that ftsh4 mutants suffer an endogenous oxidative stress. The comparative proteomic 279
analysis revealed also that, in contrast to the several enzymes of amino acid 280
metabolism to be decreased in ftsh4, isovaleryl-CoA dehydrogenase (IVDH), which is 281
involved in the catabolism of branched-chain amino acids and lysine, accumulated in 282
both ftsh4 mutants in LD, 30°C (Table I). Degradation products of these amino acids 283
can provide electrons to the respiratory chain via the ETF complex (Araújo et al., 284
2011). One can assume that in ftsh4 mutants this catabolic pathway compensates for 285
the reduced electron supply from the TCA cycle at least in LD, 30°C. 286
287
Proteins Carbonylated to a Higher Extent in ftsh4 than in Wild Type 288
289
Extensive carbonylation of mitochondrial proteins was observed in ftsh4 290
mutants growing under LD, 30°C (Fig. 2B), confirming data on plants grown at SD, 291
22°C (Gibala et al., 2009). To prove a direct and/or indirect role of FTSH4 in 292
preventing accumulation of carbonylated proteins we used a revertant line of ftsh4-1 293
(ftsh4-1-FTSH4) constitutively expressing full-length FTSH4 cDNA under the control 294
of the CaMV 35S promoter. As expected, the level of carbonylated proteins in ftsh4-295
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1-FTSH4 was comparable to that in wild type plants and the revertant plants had lost 296
the morphological aberrations characteristic for ftsh4-1 seedlings in LD, 30°C (Fig. 297
2A and Fig. 2B). 298
To identify the proteins carbonylated in the absence of FTSH4, mitochondrial 299
protein samples isolated from WT and ftsh4 plants grown under conditions inducing 300
the phenotype were first separated according to their pI, derivatized in-gel with 2,4-301
dinitrophenylhydrazine (DNPH), and then resolved by SDS-PAGE. After blotting the 302
membrane was probed with polyclonal anti-DNP-adduct antibodies (Fig. 3). A protein 303
spot was considered to be significantly increased in carbonylation level if the 304
oxidation fold of its immunological signal in ftsh4 to that in the WT gel was ≥1.2. 305
Protein spots found to be increasingly carbonylated in ftsh4 were matched to the 306
corresponding spots in an IEF/SDS-PAGE gel stained with Coomassie and identified 307
by mass spectrometry. The carbonylated proteins accumulating in ftsh4-1 under SD, 308
22°C and in ftsh4-1 or ftsh4-2 under LD, 30°C are listed in the Supplemental Table 309
S2. 310
In total, 26 mitochondrial proteins were more heavily carbonylated in ftsh4 311
than in wild type plants. They represented several functional groups located in 312
different mitochondrial compartments (Supplemental Table S2 and Supplemental Fig. 313
S3). Nine of those 26 proteins were identified in all analyzed conditions: two subunits 314
of complex V (ATP synthase subunit 1, ATP1 and ATP2), two components of the 315
TCA cycle (fumarase 1, FUM1 and succinyl Co-A ligase, SuCoA), three associated 316
with photorespiration (lipoamide dehydrogenase, MTLPD1, aminomethyltransferase, 317
GDC-T and SHM1), a stress-related protein (manganese superoxide dismutase 1, 318
MSD1), and a protease (mitochondrial processing peptidase subunit beta, 319
MPPBETA) (Supplemental Table S2). Furthermore, a number of proteins like 320
aconitase 2 (ACO2), SHM1, GDC-T, ATP1 and ATP2 were found to be more 321
extensively carbonylated in more than one spot on the 2D-OxyBlots. The different 322
spots of ACO2 and SHM1 showed differences in pI, while those of GDC-T, ATP1 and 323
ATP2 - in molecular weight; thus the last three are typical examples of protein 324
degradation products (Supplemental Table S2). 325
However, the proteins identified as carbonylated more strongly in ftsh4 than in 326
wild type plants using the OxyBlot analysis showed diverse changes in abundance as 327
determined by the 2D-DIGE assay, some being up- and some down-regulated (Table 328
I, Supplemental Table S1). Therefore, to find proteins carbonylated to a higher extent 329
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in ftsh4 than in wild type, oxidation indexes were determined for each spot of interest. 330
To calculate the oxidation index, the carbonyl immunoreactivity of each spot was 331
divided by the relative protein abundance estimated by the DIGE analysis 332
(Supplemental Table S3). Regardless of the conditions tested, ACO2 was found to 333
be more heavily carbonylated in ftsh4 compared to wild type plants (Table II, 334
Supplemental Table S3). ACO2 represents a group of proteins with a high oxidation 335
index (≥1) and a decreased protein amount estimated by DIGE (Table II, 336
Supplemental Table S3). This set of proteins includes complex I (75-kDa subunit) 337
and complex V (ATP1, ATP2, and ATP synthase subunit Fad, MGP1) subunits, TCA 338
cycle components (ACO2, SuCoA subunit beta, and malate dehydrogenase 2, 339
MDH2), enzymes of cysteine (cysteine synthase C1, ATCYSC1) and glutamate 340
(glutamate dehydrogenase 2, GDH2) metabolism, and MPPBETA. Analysis of 341
transcript level of these proteins using quantitative PCR (Fig. 4A) indicates that 342
except ATP2 other proteins did not show any decrease in mRNA expression. Thus, it 343
is conceivable that these particular proteins are extensively oxidatively damaged and 344
thus degraded in plants lacking FTSH4. In contrast, the remaining proteins with a 345
high oxidation index were found to accumulate in ftsh4 plants according to 2D-DIGE. 346
This group of proteins comprises an enzyme of the TCA cycle (FUM1), four 347
photorespiration enzymes (GDC-T, SHM1, MTLPD1, and formate dehydrogenase, 348
FDH), glutamate dehydrogenase 1 (GDH1) and a key enzyme in the elimination of 349
mitochondrial superoxide radicals, MSD1. qRT-PCR analyses indicated that 350
accumulation of these proteins is not accompanied with an increase in their transcript 351
level (Fig. 4A). Most transcripts exhibited statistically insignificant changes in ftsh4-1 352
and ftsh4-2 compared to wild type, with a log2 ratio below 0.5. Unexpectedly, the 353
transcript level of GDC-T and SHM1, proteins that accumulate in our experimental 354
conditions in the ftsh4 mutants, substantially decreased (Fig. 4A). 355
356
Increased Expression Level of ATP-dependent Proteases in ftsh4 Mitochondria 357
358
In yeast and mammals it was shown that carbonylated proteins are removed 359
by ATP-dependent proteases, presumably to avoid formation of harmful aggregates 360
(Anand et al., 2013). Therefore, we examined the expression level of known 361
mitochondrial ATP-dependent proteases in the ftsh4 mutant compared to wild type 362
plants. No significant changes were observed in the expression of mitochondrial 363
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ATP-dependent proteases in ftsh4 plants grown under LD, 22 °C, neither at the 364
transcript nor at the protein level (Fig. 5A). However, under conditions inducing the 365
phenotype, when carbonylated proteins accumulated in mitochondria of ftsh4, we 366
observed a significant increase of FTSH10 protease at the transcript and protein 367
levels, and a higher abundance of FTSH3 at the protein level (Fig. 5A, B and C). The 368
accumulation of LON1 detected by 2D-DIGE agrees with its transcriptional up-369
regulation (Fig. 5A). The abundance of other plant mitochondrial proteases (FTSH11, 370
LON4, CLP) because of a lack of corresponding antibodies was estimated only at the 371
transcript level, which did not change significantly in the ftsh4 mutant compared to 372
wild type plants (Fig. 5A). Taken together, the accumulation of carbonylated proteins 373
under conditions inducing the phenotype in ftsh4 was correlated with elevated 374
expression of at least three ATP-dependent proteases: LON1, FTSH3 and FTSH10. 375
376
ftsh4 Contains a Decreased Abundance and Activity of Complexes I and V as 377
well as an Reduced Intra-mitochondrial Pool of ATP 378
379
Previously, using a combination of blue-native polyacrylamide gel 380
electrophoresis (BN-PAGE) and histochemical staining, we have observed that loss 381
of FTSH4 was associated with a decreased abundance and activity of complexes I 382
and V in plants growing in SD, 22°C (Kolodziejczak et al., 2007). Using the same 383
approach we observed reduced amount and activity of complexes I and V in ftsh4-1 384
and ftsh4-2 mutants growing in LD, 30°C as well (Fig. 6B, C). Additionally, a slight 385
(~20%), but statistically significant decrease of activity of the analyzed complexes 386
was also detected in the ftsh4 mutants in LD at 22°C (Fig. 6A, C). Moreover, DIGE 387
analysis confirmed these results; lower amounts of NADH-ubiquinone oxidoreductase 388
75-kDa belonging to complex I, as well as ATP1 and ATP2 of complex V were found 389
in the ftsh4 mutants compared to wild type in all experimental setups (Table I). A 390
clear exception was gamma carbonic anhydrase 2 (CA2), a subunit of complex I, 391
which accumulated in the mutants. Thus, two experimental approaches, BN-PAGE 392
and 2D-DIGE, indicate that FTSH4 is essential for stability/assembly/activity of 393
complexes I and V under conditions inducing the phenotype. 394
We next tested whether this defect in the OXPHOS complexes of ftsh4 is 395
associated with a reduced intra-mitochondrial pool of ATP. Therefore, using a 396
bioluminescence assay, the ATP content was examined in mitochondria obtained 397
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from 3-week-old WT or ftsh4 plants grown under LD, 30°C. The amount of ATP in 398
mitochondria of ftsh4 was approx. 30-40% lower than in wild type (6.42 ± 2.01 for 399
WT, 3.81 ± 1.48 for ftsh4-1 and 4.3 ± 0.72 for ftsh4-2, in pmol/mg of protein; Fig. 6D). 400
This significantly reduced intra-mitochondrial pool of ATP in plants lacking FTSH4 401
protease likely reflects a perturbation in ATP formation due to a defect in 402
stability/activity of complex V. 403
404
Availability of ATP Limits Degradation Rate of Carbonylated Proteins in 405
Mitochondria of ftsh4 406
407
Given the apparent increase in abundance of ATP-dependent proteases in 408
mitochondria of ftsh4 grown at the phenotype-inducing conditions, it was surprising to 409
note that carbonylated proteins were not efficiently removed. Thus, we hypothesized 410
that in the mutants carbonylated mitochondrial proteins accumulated due to a lack of 411
ATP, required for the proteolytic activity of the ATP-dependent proteases. To test this 412
hypothesis we performed in vitro time-course experiments monitoring the level of 413
carbonylated proteins in mitochondria isolated from 2-week-old seedlings grown 414
under LD, 30°C in the absence or presence of ATP (Fig 7A). Densitometric analyses 415
indicated that the total amount of carbonylated proteins in WT mitochondria 416
significantly decreased (about 40%) when incubated without ATP for 16 h at 22°C, 417
while in ftsh4-1 and ftsh4-2 mitochondria the amount of carbonylated proteins did not 418
change significantly under the same conditions (Fig. 7A). However, in the presence 419
of ATP the amount of carbonylated proteins was strongly diminished in ftsh4 420
mitochondria incubated for 16 h at 22°C, practically exceeding the level observed in 421
wild type sample (Fig. 7A). 422
The same assay was performed on freshly isolated mitochondria from WT and 423
ftsh4 seedlings grown at LD, 22°C, incubated with succinate and antimycin A, an 424
inhibitor of complex III and a well-known inducer of oxidative stress (Fig. 7B). 425
Treatment of ftsh4 mitochondria with antimycin A for 16 h in the absence of ATP 426
resulted in a significant accumulation of carbonylated proteins, while supplementing 427
the incubation medium with ATP induced a strong decrease of oxidatively modified 428
proteins (approx. 60%, Fig. 7B). Addition of AEBSF, an inhibitor of serine proteases, 429
to the medium prevented the carbonylated protein content from decreasing in WT 430
and ftsh4-1, indicating an important contribution of serine proteases in eliminating 431
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oxidatively modified proteins in plant mitochondria (Fig. 7B). No such effect could be 432
observed using ortho-phenanthroline, an inhibitor of metalloproteases (Fig. 7B). 433
434
ftsh4 Contains Altered Mitochondrial Morphology and Phospholipid Content 435
436
Several recent findings point to an essential role of yeast and mammalian i-437
AAA proteases in the mitochondrial quality control at the organellar level (Stiburek et 438
al., 2012; Li et al., 2015; Qi et al., 2015). To look into a putative role of FTSH4 in 439
plant mitochondrial dynamics, we first examined the mitochondrial morphology of wild 440
type and ftsh4-1 plants grown under LD, 22°C (no visible phenotype) and LD, 30°C 441
(visible phenotype), transformed with a construct expressing mitochondria-targeted 442
green fluorescent protein (GFP) under the control of the CaMV 35S promoter. 443
Analysis of confocal microscopy photographs revealed the existence of a 444
heterogeneous mitochondrial population in ftsh4-1 protoplasts compared to WT (Fig. 445
8A). This heterogeneous population was characterized by the appearance of 446
enlarged, spherical mitochondria, termed “giant” mitochondria, among healthy, oval 447
forms (Fig. 8A). While the giant mitochondria were rare in ftsh4-1 protoplasts isolated 448
from plants grown at 22°C, their number and area were highly increased, when the 449
plants were exposed to the moderately elevated temperature of 30°C (Fig. 8A). In 450
addition, some of giant mitochondria displayed GFP voids, with reduced or absent 451
fluorescence signal, the feature characteristic for oxidative stress (Fig. 8A) (Logan et 452
al., 2003). Even mitochondrial enlargement was reported to be caused by oxidative 453
stress (Scott and Logan, 2008), but also by depletion of cardiolipin (CL), a signature 454
phospholipid of mitochondria (Pineau et al., 2013; Pan et al., 2014). To investigate, if 455
the aberrant mitochondrial morphology observed in ftsh4 plants is associated with 456
perturbed cardiolipin abundance, we determined the lipid composition of 457
mitochondrial membranes in WT and ftsh4 mutants grown under LD, 22°C and LD, 458
30°C using a comparative quantitative lipidomic approach (shotgun lipidomics) (Fig. 459
8B). Figure 8B shows quantification of CL and lipids involved in this phospholipid 460
biosynthesis (PA, phosphatidic acid; DAG, diacylglycerol; PI, phosphatidylinositol; 461
PG, phosphatidylglycerol) as well as the most abundant lipids of mitochondrial 462
membrane (PC, phosphatidylcholine; PE, phosphatidylethanolamine). The 463
quantitative analysis of all identified lipid classes is presented in Supplemental Fig. 464
S4. Our results indicate that the level of cardiolipin was slightly decreased in 465
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15
mitochondria of ftsh4 plants grown under LD, 22°C. This effect was more pronounced 466
under LD, 30°C, compared to wild type (Fig. 8B). Additionally, we detected an 467
accumulation of phosphatidyloglicerol (PG) in ftsh4 mitochondria under LD, 22°C, 468
which was stronger under LD, 30°C. Furthermore, the levels of phosphatidyloinositol 469
(PI) and diacyloglicerol (DAG) in ftsh4 were slightly increased (Fig. 8B). It should be 470
emphasized that DAG, PI and PG are involved in the cardiolipin biosynthesis 471
pathway, and possibly their accumulation in ftsh4 mitochondria is related to the 472
deficiency of CL; similar changes of the PG level were observed in an Arabidopsis 473
mutant disrupted in the single-copy gene encoding cardiolipin synthase (Pan et al., 474
2014). While no significant difference was observed in the amount of 475
phosphatidyloethanolamine (PE) in wild type and ftsh4 grown at LD, 22°C, ftsh4 476
plants grown at LD, 30°C had lower level of PE in their mitochondrial membranes 477
(Fig. 8B). This effect might reflect a complex regulation of the phospholipid level, 478
since both CL and PE are known to be involved in similar processes in mitochondrial 479
membranes (Joshi et al., 2012; Böttinger et al., 2012). Taken together, these results 480
demonstrate that FTSH4 protease affects the abundance of cardiolipin, and in turn 481
mitochondrial dynamics. 482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
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16
DISCUSSION 499
500
In this study, we show that loss of the FTSH4 mitochondrial protease under 501
conditions inducing the phenotype leads to oxidative damage of many mitochondrial 502
proteins different in function and submitochondrial localization. We believe that the 503
main cause of this phenotype is altered content of cardiolipin in the mitochondrial 504
membrane, associated with perturbation of at least two processes: functionality of the 505
OXPHOS system and mitochondrial dynamics. Deregulation of these processes 506
disturbs the mitochondrial quality control on molecular and organellar level, and leads 507
to the accumulation of oxidatively damaged proteins (Fig. 9). In other words, we 508
argue here that the accumulation of carbonylated proteins found in ftsh4 plants is due 509
to their more intensive generation, an ineffective system for their removal by ATP-510
dependent proteases and an altered system of mitochondrial 511
fusion/fission/mitophagy. 512
513
Diverse Mitochondrial Proteins Undergo Carbonylation in ftsh4 Grown Under 514
Conditions Inducing the Phenotype 515
516
We previously reported that ageing ftsh4 plants growing in SD, 22°C, 517
experienced oxidative stress (Gibala et al., 2009, Kicia et al., 2010). We have now 518
extended those studies by identifying the proteins to be excessively carbonylated in 519
ftsh4 and linking their oxidation status with their abundance estimated by 2D-DIGE 520
(Tables I and II). These experiments were not limited to ftsh4 growing under SD, 521
22°C, but similar analysis was also performed for ftsh4-1 and ftsh4-2 growing under 522
LD, 30°C. Diverse proteins from the OXPHOS system, TCA cycle, and 523
photorespiration, as well as manganese superoxide dismutase and cysteine synthase 524
were found to be more excessively carbonylated in the mutant than in wild type 525
mitochondria (Table II). It should be emphasized that all the oxidatively damaged 526
proteins identified in ftsh4 have been reported earlier as targets for carbonylation in 527
plant mitochondria (Kristensen et al., 2004; Smakowska et al., 2014). Those proteins, 528
which were predominantly carbonylated in the absence of FTSH4, were also 529
identified by 2D-DIGE analyses, and found to be differentially expressed compared to 530
the wild type mitochondrial proteome (Table I). A decrease in abundance was 531
observed for two enzymes of the TCA cycle (ACO2 and SuCoA), two subunits of 532
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17
complex V (ATP1 and ATP2) and the 75-kDa subunit of complex I, while fumarase 533
and two enzymes of photorespiration (GDC-T and MTLPD1) showed increased 534
abundance (Tables I and II). The lower abundance and enhanced carbonylation of 535
complex I and V subunits in mitochondria from ftsh4 were presumably caused by a 536
defect in their assembly/stability (Kolodziejczak et al., 2007). This defect may 537
increase the pool of unassembled/misfolded proteins, which will undergo 538
carbonylation and subsequent degradation. We believe that in ftsh4 the breakdown 539
of ATP1 and ATP2, ACO2 and SuCoA occurs to a certain degree after oxidation, 540
particularly since the level of their transcripts did not decrease significantly (Fig. 4). 541
The question arises as to why the carbonylated enzymes FUM1, GDC-T, 542
MTLPD1, in contrast to carbonylated ATP2 or ACO2, are present in higher 543
abundance in ftsh4 (Table II). In light of the report showing that chloroplast FTSH 544
proteases degrade functionally assembled proteins that have undergone oxidation 545
(Lindahl et al., 2000), it seems likely that FTSH4 fulfills a similar function in 546
mitochondria and its loss causes accumulation of its substrates - a specific set of 547
highly carbonylated proteins. However, there is a topological incompatibility: the 548
catalytic domain of FTSH4 protease faces the intermembrane space, while GDC-T, 549
MTLPD1 and FUM1 are matrix proteins. At this stage, we do not know how to explain 550
this observation. 551
552
ftsh4 Displays Altered Metabolic Pathways As a Response to Oxidative Stress 553
554
The ftsh4 mutant seems to provide protection against the oxidizing conditions 555
in mitochondria at several levels. Induction of non-phosphorylating alternative 556
pathways is indicated by increased transcription of the genes encoding AOX1A, 557
NDB2 and NDB4 (Fig. 4B). These pathways are known to play an essential role in 558
the response of plant mitochondria to oxidative stress mainly by preventing the 559
overreduction of the mitochondrial electron transport chain and thus the production of 560
ROS (Moller, 2001; Vanlerberghe, 2013). Furthermore, the obvious up-regulation of 561
isovaleryl-CoA dehydrogenase at the transcript and protein level (Fig. 4A, Table I) 562
suggests another non-classical entry route of electrons to the respiratory chain to be 563
activated, provided by the catabolism of branched-chain and aromatic amino acids 564
(Araújo et al., 2010). This makes sense in the light of the elevated amino acids poll 565
generated by degradation of carbonylated proteins in the ftsh4 mutant. The activation 566
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18
of this pathway by oxidative stress was reported earlier by Lehmann et al. (2009). 567
Using 2D-DIGE we further confirmed our earlier findings (Gibala et al., 2009) that 568
mitochondrial chaperones are accumulating in the ftsh4 mutant (Table I). The up-569
regulation of HSP70 and HSP60 chaperones on transcript and protein level in ftsh4 is 570
consistent with their protective roles against aggregation of mitochondrial proteins 571
under oxidative stress conditions (Table I, Fig. 4A) (Bender et al., 2011). 572
573
In ftsh4 the Defective OXPHOS System is Linked to Enhanced Generation of 574
ROS and Carbonylated Proteins 575
576
The increase in steady-state level of ROS observed in ftsh4 plants under 577
conditions inducing the phenotype (Gibala et al., 2009, Kicia et al., 2010) is leading in 578
consequence to the generation of carbonylated proteins. The trigger of the oxidative 579
stress observed in ftsh4 most likely is the dysfunctional assembly/stability/activity of 580
complex I and V under conditions inducing the phenotype (Kolodziejczak et al., 2007, 581
Fig. 6B and C). Under optimal conditions (LD, 22°C) these alterations were mild (Fig. 582
6A), but with time (late phase of vegetative growth in SD, 22°C) or under conditions 583
with a higher probability of electron leakage like moderate heat stress (LD, 30°C), 584
harmful overproduction of ROS occurs. Defects in the OXPHOS system caused by 585
the absence of FTSH4 could be explained by at least two not mutually excluding 586
mechanisms: i) The lack of chaperone-like activity of FTSH4 protease required for 587
the formation/stability of complex I and V and ii) the low level of membrane CL 588
documented in this study (Fig. 8). It is well known that CL is a structural component 589
of many protein complexes and supercomplexes of the inner mitochondrial 590
membrane in yeast, human and in plant mitochondria (Pfeiffer et al., 2003; Petrosillo 591
et al., 2007; Pineau et al., 2013; Gonzalvez et al., 2013). Reduced content of the 592
respiratory complex I/complex III supercomplex and to a lesser extent complex I was 593
found in an Arabidopsis thaliana knock-out mutant depleted in the final enzyme of the 594
cardiolipin biosynthesis (Pineau et al., 2013) 595
The results presented here suggest also that extended oxidative stress in 596
ftsh4 creates other additional sources of ROS, which further accelerate this stress. 597
Oxidatively damaged iron-sulfur proteins like aconitase or the 75-kDa subunit of 598
complex I could accelerate oxidative stress by the Fenton reaction (Moller et al., 599
2011). The finding of several mitochondrial enzymes of photorespiration being highly 600
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19
carbonylated (GDC-T, SHM1 and MTLPD1), combined with the decreased 601
abundance of other photorespiration enzymes (GLDP1 and GLDP2) suggests that 602
this process is at least slowed down in ftsh4 mitochondria (Tables I and II). Since 603
photorespiration is considered to be important to prevent ROS accumulation (Voss et 604
al., 2013), the postulated decreased activity of the GDH/SHMT complex could be an 605
additional cause for oxidative stress in ftsh4. Furthermore, among the proteins 606
carbonylated preferentially in ftsh4 is the manganese superoxide dismutase, a key 607
enzyme to eliminate mitochondrial superoxide radicals (Wang et al., 2010). This 608
enzyme is probably inactive in ftsh4 due to oxidative modification (Qin et al., 2009) 609
and it was found to be highly carbonylated in all the conditions examined (Table II). 610
611
In ftsh4 the Defective OXPHOS System is Linked to Limited ATP-dependent 612
Degradation of Carbonylated Proteins 613
614
The protease responsible to degrade oxidatively damaged matrix proteins in 615
yeast (Bender et al., 2011; Bayot et al., 2010) and mammalian mitochondria (Bota 616
and Davies, 2002) is the ATP-dependent Lon protease. However, recent studies 617
indicate in Arabidopsis the mitochondrial LON1 protease to be nonessential for the 618
turnover of oxidized proteins (Solheim et al., 2012). Nevertheless, in the absence of 619
FTSH4 we observed overexpression of mitochondrial ATP-dependent proteases, 620
potentially able to degrade oxidized proteins, at the transcriptional (LON1 and 621
FTSH10) and protein (FTSH3) levels (Fig. 5). Curiously, despite this increased 622
expression of ATP-dependent proteases, we documented that degradation of 623
carbonylated proteins in mitochondria isolated from ftsh4 was less efficient than in 624
wild type in the absence of ATP, but similar to wild type after addition of ATP (Fig. 7). 625
Using respective inhibitors we could support a previous study (Sweelove et al., 2002) 626
showing serine proteases to be mainly responsible for the degradation of oxidatively 627
modified proteins in plant mitochondria (Fig. 7). Thus, the accumulation of 628
carbonylated proteins in ftsh4 seems to be caused by limited ATP-dependent 629
proteolytic capacity in ftsh4 mitochondria. The lower ATP content found in ftsh4 630
mitochondria compared to wild type (Fig. 6D) most probably results from impaired 631
efficiency of the OXPHOS system, particularly the substantially decreased activity of 632
the ATP synthase. 633
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20
Decreased Level of CL induces Giant Mitochondria and Accumulation of 634
Carbonylated Proteins in ftsh4 635
636
One of the most exciting conclusions rationalizing the accumulation of 637
carbonylated proteins in ftsh4 is based on the observation of giant mitochondria and 638
on CL deficiency in the mitochondrial membrane in the absence of FTSH4 (Fig. 8A 639
and B). In Arabidopsis, CL deficiency has previously been linked to restricted fission, 640
which in turn causes the appearance of giant mitochondria (Pan et al., 2014). 641
Ongoing cycles of fusion and fission of mitochondrial membranes are essential for an 642
efficient defense against mitochondrial damage (Tatsuta and Langer, 2008). 643
Furthermore, in animals it was documented that giant mitochondria block mitophagy, 644
the elimination of damaged mitochondria (Zhang et al., 2014c). Thus, we postulate 645
that in mitochondria of ftsh4 carbonylated proteins are not removed efficiently, not 646
only because of the limited capacity of the ATP-dependent proteolytic system, but 647
also because of a restricted fission/mitophagy (Fig. 9). 648
It is well documented that the proteolytic activity of FTSH4 homologues in 649
yeast and mammalian is controlling the accumulation of key mitochondrial 650
phospholipids by turn-over of the phospholipid regulators (Potting et al., 2010; Potting 651
et al., 2013). In yeast, the proteins Ups1 and Ups2 in the intermembrane space act 652
as central regulators of mitochondrial phospholipid homeostasis. The turnover of 653
Ups2 is mediated by the i-AAA protease Yme1, whereas Ups1 is degraded by Yme1 654
and the metallopeptidase Atp23. Our results point to FTSH4 being a part of a 655
regulatory pathway that influences the abundance of CL in plant mitochondria. It 656
seems that this pathway is at least to some extent similar to that discovered in yeast, 657
given that in the Arabidopsis genome there is a homologue of the Ups1 protein 658
(At5g13070), while a homologue of Ups2 has not yet been found. 659
660
Conclusions 661
662
Overall, the presented results indicate that the plant protease FTSH4 663
suppresses oxidative damage in mitochondrial proteins indirectly by controlling the 664
abundance of cardiolipin, a key phospholipid within the mitochondrial membrane. 665
Availability of CL has previously been shown to stabilize respiratory complexes and 666
mitochondrial dynamics. Our findings indicate that both of these processes are 667
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21
impaired in mitochondria lacking FTSH4 and in consequence plants suffer oxidative 668
stress. Although we have been unable to identify carbonylated proteins as direct 669
substrates for FTSH4, we believe this protease could functionally resemble 670
chloroplastic FTSH in this respect (Lindahl et al., 2000), but in different 671
developmental stages and/or environmental conditions than used in our work. 672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
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22
MATERIALS AND METHODS 702
703
Plant Material and Growth Conditions 704
Arabidopsis thaliana wild type and T-DNA insertion lines were of the Columbia-0 705
(Col-0) ecotype. The transgenic lines ftsh4-1 (SALK_035107/TAIR) and ftsh4-2 706
(GABI_103H09/TAIR) were obtained from the Salk Institute and the Max Planck 707
Institute for Breeding Research, respectively. The lines were previously characterized 708
in Gibala et al. (2009). Plants were grown in growth chambers in a 16 h light/8 h dark 709
(long day, LD) photoperiod at 22°C and 30°C for 2 or 3 weeks (agar plates or 710
hydroponic culture) as well as in soil in a 8 h light/16 h dark (short day, SD) 711
photoperiod at 22°C for 9 weeks and for 4 weeks in LD at 22°C or 30°C, with a light 712
intensity of 150 μmol m-2 s-1. 713
714
Isolation of Mitochondria 715
Isolation of mitochondria from 9-week-old rosettes was performed as described by 716
Urantowka et al. (2005), and from the seedlings as described by Day et al. (1985). 717
Purified mitochondrial fractions were resuspended in a final wash buffer at a 718
concentration of 10 to 20 mg of mitochondrial protein/ml. The protein concentration 719
was determined using the DC Protein Assays (Bio-Rad, Hercules, California, USA). 720
721
Protein Leaf Extracts 722
Total protein extract was obtained from 3-week-old hydroponic cultures as described 723
by Martinez-Garcia et al. (1999). Protein concentration was determined using the DC 724
Protein Assay. 725
726
Immunoblot Analysis 727
Equal amounts of proteins from wild type (WT) and ftsh4 plants (25 μg per line) were 728
separated by SDS-PAGE according to Laemmli (1970). After electrophoresis, 729
proteins were transferred to PVDF membrane and immunostained with appropriate 730
antibodies. The antibodies used were purchased from Agrisera: anti-AtFTSH10 731
(AS07 251, Sweden) and anti-AtFTSH3 (AS07 204, Sweden) (Piechota et al., 2010). 732
Immunodetection was performed using the WesternBrightTM Quantum Western 733
Blotting Detection Kit (Advansta, Menlo Park, California, USA). The membranes were 734
documented using a chemiluminescence imager (G-BOX ChemiXR5, Syngene) and 735
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23
the optical density of the bands was quantified using the Image Quant software 736
(Molecular Dynamics, Sunnyvale, California, USA). 737
738
BN-PAGE and Catalytic Staining 739
Blue-native gel electrophoresis (BN-PAGE) was performed as described by 740
Kolodziejczak et al. (2007). The catalytic staining of mitochondrial complex I and 741
supercomplex I + III2 was carried out according to Zerbetto et al. (1997), while the 742
staining of complex V according to Van Lis et al. (2003). 743
744
Two-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE) 745
For 2D-DIGE analysis mitochondrial proteins from wild type and mutant (ftsh4-1 and 746
ftsh4-2) were precipitated with cold acetone for 2 h at -20°C and pelleted at 18000 g 747
and 4°C for 30 min. Mitochondrial protein pellets were resuspended in lysis buffer (8 748
M urea, 4% w/v CHAPS, 50 mM DTT, 40 mM Tris-HCl, pH 7.5) at a concentration of 749
10 mg of mitochondrial protein/ml and vortexed for 30 min at 4°C. The non-soluble 750
material was removed by centrifugation at 18000 g for 20 min. Each 25 μg of 751
biological replicate sample was labeled with 0.1 nmol G-Dyes (G-200 or G-300, 752
DyeAgnostics, Halle, Germany) on ice for 30 min in the dark according to 753
manufacturer’s instruction. The reaction was stopped with 1 nmol of lysine. A G-100 754
stained sample (25 μg of mitochondrial proteins), composed of equal amounts of wild 755
type and ftsh4-1 or ftsh4-2 mitochondrial protein extracts, constituted an internal 756
standard (IS) used for normalization of 2D-DIGE gels. 25 μg aliquots of each 757
differentially labeled mitochondrial sample (G-100-, G-200- and G-300-labelled 758
sample) were pooled, mixed with rehydration solution (7 M urea, 2 M thiourea, 2% 759
CHAPS, 20 mM DTT, 0.6% IPG buffer 3-11 NL (GE Healthcare, Uppsala, Sweden)) 760
and loaded on a 24-cm, pH 3-11 NL Immobiline DryStrip (GE Healthcare). 761
Rehydration of the strips and first dimension electrophoresis (isoelectric focusing, 762
IEF) were conducted in an Ettan IPGphor Isoelectric Focusing System (GE 763
Healthcare) at 50 V for 12 h (rehydration), 500 V for 3 h (step), 2000 V for 2 h (step), 764
8000 V for 1 h (gradient), 8000 V for 10 h (step) at a maximum setting of 50 μA per 765
strip. After IEF, the strips were equilibrated for 15 min in the equilibration buffer (6 M 766
urea, 30% glycerol, 50 mM Tris-HCl (pH 8.8.), 2% w/v SDS, trace of bromophenol 767
blue) supplemented with 65 mM dithiothreitol and then for a further 15 min in the 768
same buffer with 135 mM iodoacetamide. The strips were laid on top of 12.5% w/v 769
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24
polyacrylamide gels (26 x 20 cm) and sealed with 0.5% agarose in 25 mM Tris-HCl, 770
192 mM glycine, 0.2% SDS. Second dimension electrophoresis was performed 771
overnight in an Ettan Dalt II electrophoresis unit (GE Healthcare) at 20°C and 1 W 772
per gel. After electrophoresis the gels were scanned with a Typhoon 9400 scanner 773
(GE Healthcare) at the excitation wavelengths corresponding to each of the G-Dyes. 774
The images of the gels from three independent biological repetitions were analysed 775
using DeCyder software version 6.5 (GE Healthcare) for normalization and statistical 776
analysis. Protein spots that showed a significant difference in abundance (fold 777
difference ±1.2, p 0.05) between ftsh4 and wild type were picked from the gel and 778
identified by MALDI-TOF (PMF, Peptide Mass Fingerprinting). In this approach three 779
experimental setups were analyzed (SD, 22°C for ftsh4-1; LD, 30°C for ftsh4-1 and 780
LD, 30°C for ftsh4-2) in three independent biological replicates. 781
782
Detection of Carbonylated Proteins on One-Dimensional gels 783
Carbonylated proteins were detected and analyzed following derivatization of protein 784
carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH), using the OxyBlot kit 785
(Millipore, Billerica, California, USA). Immunodetection was performed with a primary 786
antibody directed against dinitrophenylhydrazone using 25 μg of total protein extract 787
or 30 μg mitochondrial proteins from ftsh4 and WT plants per lane. The carbonylated 788
proteins were visualized with the WesternBrightTM Quantum Western Blotting 789
Detection Kit. 790
791
Two-Dimensional OxyBlots 792
Mitochondrial protein extracts (150-200 μg) of wild type, ftsh4-1 and ftsh4-2 lines 793
were prepared as described above. After IEF, the strips were frozen at -80°C for 2 h. 794
Derivatization of protein carbonyl groups was done by incubation of the strips in a 795
buffer containing 2 M HCl and 10 mM 2,4-dinitrophenylhydrazine for 20 min at room 796
temperature. Derivatized proteins were neutralized by repeated incubation of the 797
strips in a solution of 2 M Tris base and 30% glycerol (v/v) for 20 min at room 798
temperature. Afterwards, the strips were equilibrated and proteins were resolved on 799
second-dimension gels as described above. After electrophoresis, proteins were 800
electrotransferred onto a PVDF membrane (Bio-Rad) and subjected to 801
immunodetection of carbonyl groups using the OxyBlot kit (Millipore). 802
Immunodetection was performed with a primary antibody directed against 803
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25
dinitrophenylhydrazone and the Amersham ECL Prime Western Blotting Detection 804
system (GE Healthcare). 805
Quantitative Analysis of Two-Dimensional OxyBlots 806
Protein spots on two-dimensional OxyBlots were quantified using ImageJ Fiji 807
software (Fiji). For different experimental setups and type of sample (WT, ftsh4-1 and 808
ftsh4- 2), each spot was quantified and normalized to the total background intensity 809
of each OxyBlot. Then, the ftsh4/WT oxidation ratio was calculated for each spot. 810
Standard deviation (SD) was calculated and statistical significance was assessed 811
using Student’s t-test for three independent experiments (n=3) for each experimental 812
setup (ftsh4-1/WT, LD, 30°C; ftsh4-2/WT, LD, 30°C; ftsh4-1/WT, SD, 22°C). 813
Oxidation indexes were calculated as the ratio between oxidation fold and protein 814
abundance fold (oxidation fold/protein fold) obtained by 2D-DIGE for each spot of 815
interest. 816
817
Mass Spectrometry and Protein Identification 818
For protein identification, 150 μg of protein sample consisting of equal amounts of 819
wild type and ftsh4 mitochondrial protein extracts was run on preparative SDS gels. 820
The gels were stained with colloidal Coomassie and protein spots showing significant 821
difference in abundance between samples were manually or automatically excised 822
from the gels with an Ettan Spot Picker (GE Healthcare). Gel pieces were washed 823
briefly with deionized water and incubated for 1 h in a solution of 35% acetonitrile 824
(ACN) and 20 mM NH4HCO3. Two further washes (each 10 min) were performed with 825
100% ACN to dehydrate the gels. In-gel protein digestion was performed with 5 ng/μl 826
trypsin (Promega V5111, Madison, USA) in 20 mM NH4HCO3/10% ACN overnight at 827
37°C. The peptides were extracted with 1% trifluoroacetic acid (TFA) for 30 min at 828
room temperature and occasional shaking. A volume of 1 μl of peptide mixture was 829
spotted on an ABI-PerSpective Voyager DE STR MALDI plate (Applied Biosystems, 830
Foster City, California, USA) covered with 1 μl of alpha-cyano-4-hydroxycinnamic 831
acid (HCCA) as a matrix in a solution of 50% ACN and 0.1% TFA. Peptide mass 832
fingerprints (PMFs) of the samples were acquired using a Voyager-DE STR MALDI-833
TOF (Applied Biosystems) mass spectrometer. Peptide calibration standard mixture 834
in the mass range 800-4000 Da (Sequazyme, Applied Biosystems Siex) was used for 835
calibration of the mass spectrometer. The PMFs were searched against the 836
Arabidopsis Information Resource (TAIR) database. The fixed and variable 837
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26
modifications were cysteine carbamidomethylation and methionine oxidation, 838
respectively, with the maximum number of missed cleavages of 2. Peptide mass 839
precision tolerance error was set to 100 ppm. Proteins were identified with the 840
VoyagerTM 5 Software (Applied Biosystems) using the Mascot 2.3 search engine 841
(Matrix science, www.matrixscience.com). 842
843
RNA Isolation and cDNA Synthesis 844
Total RNA was isolated from 3-week-old hydroponically grown seedlings and rosettes 845
of plants grown in soil for approximately 9 weeks using the GeneMATRIX Universal 846
RNA Purification Kit (EURx, Gdansk, Poland). The reverse transcription reaction was 847
performed using up to 2 μg of total RNA and a reverse transcription kit (Applied 848
Biosystems). Resulting cDNA was used as a template for quantitative real-time PCR. 849
850
Real-Time PCR Analysis 851
Real-time PCR analyses were performed in a LightCycler 2.0 instrument (Roche 852
Applied Science, Mannheim, Germany). Real-Time 2x PCR Master Mix SYBR 853
version B (A&A Biotechnology, Gdynia, Poland) was used. Reactions were carried 854
out in a total volume of 15 μl with a final concentration of 0.5 μM primers. Material 855
from wild type plant served as the calibrator, and the PP2AA3 (protein phosphatase 856
2A subunit A3) gene (At1g13320) was used as a reference. The values of 857
amplification efficiency of the analysed amplicons were calculated based on standard 858
curves generated for serial 2-fold dilutions of the cDNA samples. The amplification 859
protocol comprised: denaturation, 95°C for 1 min; amplification, 45 cycles at 95°C for 860
10 s, 55-65°C (the annealing temperature was specific for primers used) for 10 s, 861
72°C for 20 s with single data acquisition; cooling, 40°C for 30 s. The specificity of 862
the amplification products was verified by melting curve analysis. The primers used 863
are listed in Supplemental Table S4. 864
865
Plasmid Construction 866
The original FTSH4 cDNA was cloned in pTZ57 R/T vector (ThermoFisher Scientific, 867
Carlsbad, Massachusetts, USA), sequenced and recloned into pENTRTM/D-TOPO® 868
(ThermoFisher Scientific) using primes described in Supplemental Table S4. In the 869
second step of cloning by the Gateway method FTSH4 was introduced under CaMV 870
35S promoter to the destination vector pGWB514 (a kind gift from Dr. Tsuyoshi 871
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27
Nakagawa, Nakagawa et al., 2007) containing the HA-tag at the C-terminus. The final 872
construct was subjected to the complementation analysis. 873
874
Agrobacterium-Mediated Transformation of A. thaliana 875
The pGWB514 plasmid was introduced into Agrobacterium tumefaciens strain 876
LBA4404 by electroporation. The obtained bacterial strain was used for the floral dip 877
vacuum infiltration of the ftsh4-1 mutants as described by Desfeux et al. (2000). 878
Transformants were checked for complementation of the developmental defects 879
under appropriate conditions. 880
881
Lipidomic analysis 882
Lipid extraction, mass spectrometric analysis and data analysis were done by 883
Lipotype GmbH (Germany). For shotgun lipidomics, lipids were extracted with 884
chloroform and methanol from either mitochondria isolated from WT, ftsh4-1 and 885
ftsh4-2 2-week-old seedlings or leaf homogenates as described in Sampaio et al. 886
2011. Samples were spiked with known amounts of lipid class-specific internal 887
standards prior to extraction and lipid extracts were subjected to mass spectrometric 888
analysis. Mass spectra were acquired on a hybrid quadrupole/Orbitrap mass 889
spectrometer (Q-Exactive, ThermoFisher Scientific) equipped with an automated 890
nano-flow electrospray ion source (Triversa Nanomate, Advion, Ithaca, New York, 891
USA) in both positive and negative ion mode. Lipid identification using Lipotype 892
Xplorer was performed as previously described (Herzog et al. 2011; Herzog et al. 893
2012). Precursor ion intensity values from mass spectra were normalized to 894
intensities of their respective internal standards to obtain pmol values. These values 895
were converted to mol% (mole fraction), to show the stoichiometric relationship 896
between lipids. 897
898
Plant Protoplast Isolation and Confocal Imaging 899
Protoplasts were isolated from leaves of 4-week-old WT and ftsh4-1 plants grown in 900
soil under LD, 22°C and LD, 30°C, and expressing mitochondria-targeted green 901
fluorescent protein (GFP) under the control of the CaMV 35S promoter, using the 902
method described by Yoo et al. (2007). In order to target GFP into mitochondria, the 903
N-terminal targeting sequence from the gene encoding mitochondrial F1F0 ATP 904
synthase delta subunit was used (Sakamoto and Hoshino, 2004). The images of 905
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28
protoplasts were acquired using a Zeiss LSM510 confocal laser scanning microscope 906
equipped with a 40x water immersion objective. The GFP signal was visualized with 907
a 488-nm argon laser and a BP505-530 filter. 908
909
In Vitro Mitochondrial Protein Degradation Assay 910
To test the rate of degradation of carbonylated proteins in mitochondria of WT and 911
ftsh4 mutants, the freshly isolated mitochondria (30 µg) from 2-week-old seedlings 912
grown in LD, 30°C on agar plates were resuspended in a washing buffer (0.3 M 913
sucrose, 10 mM TES, pH 7.5) and incubated for 16 h at 22°C with or without 3.5 mM 914
ATP. In order to induce an oxidative stress, mitochondria obtained from seedlings 915
grown in LD, 22°C were resuspended in a washing buffer containing 8 µM antimycin 916
A and 5 mM succinate and incubated for 16 h at 22°C in the presence or absence of 917
3.5 mM ATP. For protease inhibitor assays, the inhibitors of serine proteases (2 mM 918
AEBSF, Sigma, St. Louis, USA) and metalloproteases (25 mM ortho-phenanthroline, 919
Sigma) were added into the incubation medium and the mitochondrial protein 920
samples were incubated under the same conditions. After the incubation, the 921
samples were centrifuged for 10 min at 21000 g and 4°C and the mitochondrial 922
pellets were further analysed for protein carbonylation by immunodetection with anti 923
DNP-antibodies using the OxyBlot kit (Millipore). The densitometry analyses of the 924
carbonylated proteins were performed using ImageJ Fiji software (Fiji). 925
926
Measurement of Mitochondrial ATP 927
The ATP amounts in mitochondria of WT and ftsh4 plants were calculated from an 928
ATP standard curve using the ATP Determination Kit (ThermoFisher Scientific), 929
following the manufacturer’s instructions. The measured mitochondrial ATP content 930
was expressed as pmol/mg of mitochondrial protein. Mitochondria were isolated in 931
sterile conditions from 3-week-old seedlings grown in LD, 30°C. Statistical analysis of 932
differences between the ATP amounts was performed by unpaired two-tailed Student 933
t-test. 934
935
936
937
938
939
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29
Supplemental Data 940
The following materials are available in the online version of this article. 941
942
Supplemental Figure S1. Proportion, number and functional categories of 943
mitochondrial proteins differing in abundance between ftsh4 and wild type 944
Arabidopsis plants growing under different conditions inducing the phenotype. 945
946
Supplemental Figure S2. Representative differential 2D IEF/SDS-PAGE gel of 947
ftsh4-1 versus wild type mitochondrial proteins from plants grown in LD, 30°C. 948
949
Supplemental Figure S3. Proportion, number and functional categories of identified 950
mitochondrial carbonylated proteins in the ftsh4 mutants. 951
952
Supplemental Figure S4. Total lipid content and classes in mitochondria of WT, 953
ftsh4-1 and ftsh4-2 grown under optimal (22°C) and moderately elevated temperature 954
(30°C) determined by mass spectrometry (Shotgun lipidomics). 955
956
Supplemental Table S1. Identification of proteins differentially abundant in ftsh4-1 957
and ftsh4-2 in comparison to WT in 2D-DIGE. 958
959
Supplemental Table S2: Identification of carbonylated proteins accumulating in 960
ftsh4-1 and ftsh4-2 in comparison to WT in 2D-OxyBlot analysis. 961
962
Supplemental Table S3. Protein abundance, oxidation folds and oxidation indexes 963
estimated for all tested experimental setups (LD, 30°C for ftsh4-1, ftsh4-2 and SD, 964
22°C for ftsh4-1). 965
966
Supplemental Table S4. Primers used for qRT-PCR. 967
968
Supplemental Table S5. Primers used for plasmid construction, cloning and 969
genotyping. 970
971
972
973
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30
Acknowledgements 974
975
We thank Dr. Harald Aigner (Dept. of Chemistry, Umeå University, Sweden) for help 976
in designing and performing 2D-DIGE analyses. 977
978
We thank Dr. Michal Surma (Lipotype GmbH, Dresden) for help, advice and 979
discussion concerning lipidomic analysis. 980
981
The Proteomics Facility of the Chemical Biological Centre of Umeå University 982
provided the facilities for performing the 2D-DIGE experiment and the MALDI-TOF 983
mass spectrometry analyses. 984
985
986
987
988
989
990
991
992
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31
Table I. Comparative DIGE analysis of the Arabidopsis ftsh4 mitochondrial proteomes. Protein spots with abundances 993 differing between ftsh4 and WT were identified by PMF. 994 995
Protein spot
number
Accession
number (TAIR)
Name of the protein
Statistical analysis
2D-DIGE LD 30°C
2D-DIGE SD 22°C
OXPHOS
ftsh4-1/ WT*
p value ftsh4-2/ WT*
p value ftsh4-1/ WT*
p value
Complex I 1 ATMG00510.1 NADH dehydrogenase subunit 7 (NAD7)
[2.03] [0.02] 1.48 0.04
2 AT5G37510.1 NADH-ubiquinone oxidoreductase subunit 75 kDa -1.5 0.03 -1.67 0.01 -3.04 0
3 AT1G47260.1 Gamma carbonic anhydrase 2 (CA2) 2.08 0 2.78 0.04 1.98 0.01
4 AT5G66510.1 Gamma carbonic anhydrase 3 (CA3) -1.44 0.04 -1.29 0.02
ATP synthase 5 ATMG01190.1 ATP synthase subunit alpha (ATP1)
-1.6 0 -1.49 0.02 -2.01 0.02
6 AT5G08670.1 AT5G08690.1 AT5G08680.1
ATP synthase subunit beta (ATP2)
-1.26
0.01
-1.48
0.02
-3.39
0
7 AT5G08670.1 AT5G08690.1 AT5G08680.1
ATP synthase subunit beta (ATP2) [-1.27] [0.1] -1.93 0.01
8 AT5G08670.1 AT5G08690.1 AT5G08680.1
ATP synthase subunit beta (ATP2) [-1.5] [0] -1.54 0.03 -1.26 0
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9 AT5G08670.1 AT5G08690.1 AT5G08680.1
ATP synthase subunit beta (ATP2) -1.27 0.11
10 AT3G52300.1 ATP synthase D subunit (ATPQ) -1.48 0.04 -1.67 0
11 AT2G21870.1 ATP synthase subunit Fad (MGP1) -1.6 0.01 [-1.45] [0.05] [-2.7] [0.01]
TCA cycle 12 AT2G44350.1 Citrate synthase (ATCS)
-1.04 0.05 -1.32 0.01 -1.43 0
13 AT4G26970.1 Aconitase 2 (ACO2) [-1.65] [0.03] -4.85 0 -2.94 0.02
14 AT5G03290.1 NAD+-dependent isocitrate dehydrogenase V (IDH-V) -1.43 0.03
15 AT5G65750.1 2-oxoglutarate dehydrogenase E1 subunit [-1.45] [0.03] -1.65 0.03
16 AT2G20420.1 Succinyl-CoA ligase beta subunit -1.34 0.04 -1.46 0.03 1.26 0.01
17
AT5G66760.1 Succinate dehydrogenase flavoprotein subunit (SDH1-1) 1.96 0.01 1.37 0.04 14.51 0.01
18 AT2G47510.1 Fumarase 1 (FUM1) 1.59 0.01 1.3 0.05 1.02 0.01
19 AT3G15020.2 Malate dehydrogenase 2 (MDH2) -1.13 0.04 -1.3 0.02
TCA cycle adjacent 20 AT5G18170.1 Glutamate dehydrogenase 1 (GDH1)
1.87 0.01 -1.08 0.04
21 AT5G07440.1 Glutamate dehydrogenase 2 (GDH2) -1.34 0.01 [-1.12] [0.01]
22 AT3G22200.1 Gamma-aminobutyrate transaminase (GABA-T) -1.45 0.04 [-1.25] [0.85]
Photorespiration and one carbon metabolism 23 AT4G33010.1 Glycine decarboxylase P-protein 1 (GLDP1) [-1.87] [0.01] [-1.32] [0.01] -3.21 0
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24 AT2G26080.1 Glycine decarboxylase P-protein 2 (GLDP2)
-1.3 0.05 -1.04 0 -2.94 0.02
25 AT1G48030.1 Lipoamide dehydrogenase (MTLPD1) 1.2 0.05 1.43 0
26 AT1G11860.1 Aminomethyltransferase (GDC-T) 1.36 0.02 1.25 0.06
28 AT4G37930.1 Serine transhydroxymethyltransferase 1 (SHM1) -2.85 0
29 AT4G37930.1 Serine transhydroxymethyltransferase 1 (SHM1) 1.66 0.03 1.15 0.05 [-1.32] [0.04]
30 AT4G37930.1 Serine transhydroxymethyltransferase 1 (SHM1) 1.22 0.02 1.13 0.04
31 AT5G14780.1 Formate dehydrogenase (FDH) [1.45] [0.11] 2.14 0.01
Amino acid metabolism 32 AT3G45300.1 Isovaleryl-CoA -dehydrogenase (IVDH)
1.3 0.04 1.42 0.05
33 AT5G62530.1 Pyrroline-5-carboxylate dehydrogenase (P5CDH) -1.5 0 [-1.26] [0]
34 AT3G61440.1 Cysteine synthase C1 (ATCYSC1) -1.18 0 -1.07 0.09 -1.47 0
35 AT3G59760.3 O-acetylserine(thiol)lyase (OAS-C) -1.82 0.04 [-1.94] [0.04]
36 AT4G08870.1 Arginine amidohydrolase 2 (ARGAH2) -2.15 0.01
Chaperones and stress-related 37 AT3G13860.1 Heat shock protein 60-3A (HSP60-3A)
3.2 0.01
38 AT2G33210.1 Heat shock protein 60-2 (HSP60-2) 1.63 0.05 [1.25] [0.02] 1.42 0.01
39 AT2G33210.1 Heat shock protein 60-2 (HSP60-2) 1.38 0.43
40 AT4G37910.1 Heat shock protein 70-1 (mtHSP70-1) 1.62 0.02 [1.6] [0]
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41 AT5G09590.1 Heat shock protein 70-2 (mtHSP70-2)
1.12 0.01 1.48 0.02 1.89 0
42 AT5G40770.1 Prohibitin 3 (PHB3) 1.09 0 1.74 0.04 1.72 0
43 AT3G10920.2 Manganese superoxide dismutase 1 (MSD1) 1.03 0.05 1.29 0.01 1.23 0.04
Proteases 44 AT5G26860.1 LON protease 1 (LON1)
1.56 0.02
45 AT1G51980.1 Probable mitochondrial processing peptidase subunit alpha-1 (Alpha MPP1) -1.31 0.02 [-1.56] [0.01] -1.21 0.02
46 AT3G02090.1 Mitochondrial processing peptidase subunit beta (MPPBETA) -1.01 0.04 -1.57 0.02 -1.02 0.05
Transport 47 AT5G15090.1 Voltage-dependent anion-selective channel 3 (VDAC3)
3.35 0 1.46 0.01
48 AT5G67500.1 Voltage-dependent anion-selective channel 2 (VDAC2) [1.53] [0] 1.42 0.03
49 AT3G01280.1 Voltage-dependent anion-selective channel 1 (VDAC1) 1.57 0.05 1.7 0
Miscellaneous 51 AT3G48000.1 Aldehyde dehydrogenase (ALDH2B4)
-1.52 0
52 AT4G11010.1 Nucleoside diphosphate kinase 3 (NDPK3) -1.25 0.01
53 AT5G63400.1 Adenylate kinase (ADK1) -1.79 0.01
54 AT4G02930.1 Elongation factor Tu (EF-Tu) -1.27 0.03
55 AT3G07480.1 Electron carrier/ iron-sulfur cluster binding; ferredoxin-like 1.84 0.03
Contaminations
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56 AT3G01500.1 Carbonic anhydrase 1 (CA1) (chloroplast) -3.14 0 1.22 0.05
57 AT2G33150.1 Peroxisomal 3-ketoacyl-CoA thiolase 3 (PKT3) (peroxisome. glyoxisome) -2.08 0.01 -2.94 0.05
58 AT2G13360.1 Alanine:glyoxylate aminotransferase (AGT) (peroxisome) 2.94 0.05
59 AT1G20620.1 Catalase 3 (CAT3) (peroxisome) 2.22 0 1.79 0.01
60 AT5G14920.1 Gibberellin-regulated family protein (extracellular) -2.55 0 1.89 0.04
61 AT1G78850.1 Curculin-like (mannose-binding) lectin family protein (extracellular) 1.57 0.05
62 AT4G32900.1 Aminoacyl-tRNA hydrolase (cytosol) -1.33 0.03
63 AT5G45150.1 RNase THREE-like protein (RTL3) (nucleus) -1.45 0.03
64 AT2G20630.2 Protein phosphatase 2C (PP2C) (cytosol) -1.65 0.03
65 AT2G26430.1 Arginine -rich cyclin 1 (RCY1) (nucleus) -1.27 0.03
66 AT5G60240.1 Unknown protein (nucleus) -4.05 0
67 AT1G14060.1 GCK domain-containg protein (nucleus) -3.81 0
68 AT3G16420.1 PYK10-binding protein 1 (PBP1) 1.45 0
69 AT1G17290.1 Alaninine aminotransferase (ALAAT1) (cytosol) -1.38 0.04 [-1.34] [0] -1.64 0
70 AT2G29420.1 Glutathione S-transferase U7 (ATGSTU7) (cytosol) -1.04 0.06 -1.87 0.04
996 *fold change in abundance of ftsh4/WT with cut-off 1.2 and t-test p value (p≤0.05). Values not passing this cut-off are written in 997
italics, values in [ ] correspond to spots only identified based on their positions in 2D-DIGE gels due to technical limitations. 998
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Table II. Average oxidation indexes (ftsh4/WT) for selected mitochondrial proteins in all tested experimental setups (A, LD, 30°C for ftsh4-1; B) 1 ftsh4-2, and C) SD, 22°C for ftsh4-1). Spot quantification (percentage volume of each spot) was performed on 2D-OxyBlots from WT and ftsh4 2 mitochondria using ImageJ Fiji software and on 2D-DIGE gels using DeCyder 2D 6.5 software. The oxidation fold was normalized by the 3 corresponding protein fold to assess the oxidation index. Columns from left show: protein spot number on 2D-OxyBlot, accession number (TAIR), 4 protein name annotation and oxidation indexes. 5 A) LD/30°C ftsh4-1/WT B) LD/30°C ftsh4-2/WT C) /SD/22°C ftsh4-1/WT 6
7 Protein
spot number
Accession number (TAIR)
Name of the protein Oxidation Indexes SD/22°C
ftsh-1/WT 14 AT4G26970.1 Aconitate hydratase 2
(ACO2) 14.62
15 AT4G26970.1 Aconitate hydratase 2 (ACO2) 11.99
28 AT3G61440.1 Cysteine synthase (ATCYSC1) 9.4
1 AT5G37510.2 NADH dehydrogenase (ubiquinone). 75 kDa
subunit 7.21
22 AT4G37930.1 Serine
hydroxymethyltransferase 1 (SHM1)
6.08
26 AT5G18170.1 Glutamate
dehydrogenase 1 (GDH1)
5.5
24 AT1G48030.1 Mitochondrial lipoamide
dehydrogenase 1 (MTLPD1)
4.28
17 AT2G47510.1 Fumarase (FUM1) 4.15
7 ATMG01190.1 ATP synthase subunit alpha (ATP1) 3.49
19 AT1G11860.1 Aminomethyltransferase (GDC-T) 3.41
16 AT2G20420.1 Succinyl-CoA ligase subunit beta 2.95
21 AT4G37930.1 Serine
hydroxymethyltransferase 1 (SHM1)
2.71
8 AT5G08670.1 ATP synthase subunit beta (ATP2) 2.41
25 AT5G14780.1 Formate dehydrogenase (FDH) 1.82
5 AT3G02090.1 Mitochondrial processing peptidase subunit beta
(MPPBETA)1.66
9 AT5G08670.1. AT5G08690.1. AT5G08680.1
ATP synthase subunit beta (ATP2) 1.63
29 AT3G10920.2 Manganese superoxide dismutase 1 (MSD1) 1.23
Protein spot
number Accession
numer (TAIR) Name of the protein Oxidation Indexes
29 AT3G10920.2 Manganese superoxide dismutase 1 (MSD1) 17.69
24 AT1G48030.1 Mitochondrial lipoamide
dehydrogenase 1 (MTLPD1)
8.29
5 AT3G02090.1 Mitochondrial processing peptidase subunit beta
(MPPBETA) 7.29
20 AT1G11860.1 Aminomethyltransferase (GDC-T) 6.33
17 AT2G47510.1 Fumarase (FUM1)
5.68
19 AT1G11860.1 Aminomethyltransferase (GDC-T) 5.11
18 AT3G15020.2 Malate dehydrogenase (MDH2) 4.95
16 AT2G20420.1 Succinyl-CoA ligase subunit beta 4.32
21 AT4G37930.1 Serine
hydroxymethyltransferase 1 (SHM1)
4.02
6 ATMG01190.1 ATP synthase subunit alpha (ATP1) 3.47
10 AT2G21870.1 ATP synthase subunit Fad (MGP1) 2.72
9 AT5G08670.1. AT5G08690.1. AT5G08680.1
ATP synthase subunit beta (ATP2) 2.13
26 AT5G18170.1 Glutamate dehydrogenase 1 (GDH1) 1.76
28 AT3G61440.1 Cysteine synthase (ATCYSC1) 1.72
13 AT2G44350.1 Citrate synthase (ATCS)
1.58
4 AT5G66760.1 Succinate dehydrogenase
flavoprotein subunit (SDH1-1)
1.09
2 AT1G47260.1 Gamma carbonic
anhydrase 2 (CA2)
1
Protein spot
number Accession
number (TAIR) Name of the protein Oxidation Indexes
14 AT4G26970.1 Aconitate hydratase 2 (ACO2) 14.4
8 AT5G08670.1 ATP synthase subunit beta (ATP2) 8.35
15 AT4G26970.1 Aconitate hydratase 2 (ACO2) 6.67
9 AT5G08670.1. AT5G08690.1. AT5G08680.1
ATP synthase subunit beta (ATP2) 4.41
5 AT3G02090.1 Mitochondrial processing peptidase subunit beta
(MPPBETA) 4.02
1 AT5G37510.2 NADH dehydrogenase (ubiquinone). 75 kDa
subunit 3.83
21 AT4G37930.1 Serine
hydroxymethyltransferase 1 (SHM1)
3.29
6 ATMG01190.1 ATP synthase subunit alpha (ATP1) 3.21
22 AT4G37930.1 Serine
hydroxymethyltransferase 1 (SHM1)
2.96
29 AT3G10920.2 Manganese superoxide dismutase 1 (MSD1) 2.73
17 AT2G47510.1 Fumarase (FUM1) 2.61
7 ATMG01190.1 ATP synthase subunit alpha (ATP1) 2.53
16 AT2G20420.1 Succinyl-CoA ligase subunit beta 2.32
24 AT1G48030.1 Mitochondrial lipoamide
dehydrogenase 1 (MTLPD1)
1.76
19 AT1G11860.1 Aminomethyltransferase (GDC-T) 1.67
30 AT5G15090.1 Voltage-dependent anion-
selective channel 3 (VDAC3)
1.63
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Figure legends 8
9
Figure 1. Morphology of ftsh4 and WT plants growing under LD, 30°C. A, 2-week-old 10
WT and ftsh4 plants grown on agar plates. B, Delay time (in days) in leaf emergence 11
of ftsh4-1 and ftsh4-2 compared to WT. Plants were staged as described by Boyes et 12
al. (2001). C, Rosette diameter of plants grown in soil at indicated time points after 13
sowing. Mean values ± SD from three measurements are shown. Significant 14
differences are indicated by asterisks (one-sample t-test *p<0.05). D, 5-week-old 15
rosette leaves of plants grown in soil. 16
17
Figure 2. A, Morphology of 2-week-old WT, ftsh4-1 and ftsh4-1-FTSH4 (revertant) 18
seedlings grown under LD, 30°C on agar plates. B, Carbonylated proteins in a total 19
protein extract of the above genotypes. Immunodetection with anti-DNP antibodies 20
and quantification of carbonylated proteins in total protein extract, separated by one-21
dimensional gel electrophoresis. Anti-DNP signals from entire lanes were quantified 22
densitometrically. In each experiment, the values for the relative carbonylated protein 23
amount were calculated as a percentage of the value determined for the wild type 24
plants (set to 100%). Mean values ± SD from at least three independent experiments 25
are shown. Statistically significant differences in abundance between WT, ftsh4-1 and 26
ftsh4-1-FtsH4 plants are indicated by asterisks (one-sample t-test *p<0.05). 27
28
Figure 3. Comparison of mitochondrial carbonylated proteins from WT (left) and 29
ftsh4-1 (right) plants growing in LD, 30°C. Proteins separated by IEF/SDS two-30
dimensional gel electrophoresis were transferred on PVDF membrane to 31
subsequently detect carbonylated proteins using the OxyBlot technique. Arrowheads 32
refer to protein spots accumulating in ftsh4 mitochondria, which are identified in all 33
tested setups. Protein spots are listed in Table II and Supplemental Table S2. 34
35
Figure 4. Relative transcript levels for selected genes encoding mitochondrial 36
proteins in ftsh4-1 and ftsh4-2 mutants growing in LD, 30°C compared to wild type 37
plants. A, The level of transcripts for genes encoding proteins identified by DIGE 38
analysis. B, The level of transcripts for genes encoding proteins usually up-regulated 39
by oxidative stress. Relative abundance of transcripts is expressed as log2 ratios. 40
Mean values ± SD from at least three independent experiments are shown. The 41
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38
dotted lines indicate the cut-off value +/- 0.5 (log2) of the ratio corresponding to the 42
threshold level for significant up- and down-regulation of the transcripts in ftsh4. Full 43
names of genes are given in Table I and Supplemental Table S1. 44
45
Figure 5. A, Relative transcript levels for genes encoding mitochondrial ATP-46
dependent proteases in ftsh4-1 and ftsh4-2 mutants grown in LD, 22°C and LD, 30°C 47
compared to wild type plants. The relative abundance of transcripts is expressed as 48
log2 ratios. Mean values ± SD from at least three independent experiments are 49
shown. The dotted lines indicate the cut-off value +/- 0.5 (log2) of the ratio 50
corresponding to the threshold level for significant up- and down-regulation of the 51
transcripts in ftsh4. B, Representative images of immunodetection of selected 52
mitochondrial proteases in ftsh4-1 mutant compared to wild type plants growing in 53
LD, 22°C, LD, 30°C or SD, 22°C. C. Densitometric quantification of immunoblots 54
presented in B. Intensity of bands was estimated using Image Quant software 55
(Molecular Dynamics). Data for ftsh4-1 are expressed as percentage of the value for 56
wild type plants. Mean values ± SD from at least three experiments are shown. 57
Significant differences in abundance between WT and ftsh4-1 mutant are indicated 58
by asterisks (one-sample t-test *p<0.05). 59
60
Figure 6. Amounts and activities of respiratory complexes (A,B,C) and the level of 61
ATP (D) in ftsh4 and WT plants. Mitochondria were isolated from 3-week-old WT and 62
mutant plants (ftsh4-1 and ftsh4-2) growing hydroponically in LD, 22°C and LD, 30°C. 63
A, B Coomassie brilliant blue (CBB) and in-gel activity staining of complex I (CI) and 64
V (CV) after blue-native polyacrylamide gel electrophoresis. C, Quantification of 65
activities of complexes I and V. The intensity of bands was estimated by 66
densitometric analysis using Image Quant software (Molecular Dynamics). Relative 67
complex activity from mutant mitochondria was calculated as percentage of that in 68
WT plants. Differences in activity between WT and mutants are in all cases 69
statistically significant (in one-sample t-test p<0.05). Mean values ± SD from three 70
experiments are shown. D, The ATP content in WT and ftsh4 mitochondria. 71
Mitochondria were isolated from 3-week-old WT, ftsh4-1 and ftsh4-2 seedlings grown 72
under LD, 30°C. The ATP concentration was determined as described in Materials 73
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39
and Methods. The unpaired t-test was used to estimate the p values: *p<0.05. Error 74
bars correspond to SD (n=6). 75
76
Figure 7. In vitro carbonylated protein degradation in the WT and ftsh4 mitochondria. 77
Immunodetection of carbonylated proteins separated by one-dimensional gel 78
electrophoresis was estimated with anti-DNP antibodies. Anti-DNP signals from 79
entire lanes were quantified densitometrically. Mean values ± SD from three 80
experiments are shown. A, Mitochondria were isolated from 2-week-old WT, ftsh4-1 81
and ftsh4-2 seedlings grown under LD, 30°C and incubated at 22°C for 16 hours in 82
the absence or presence of 3.5 mM ATP. The unpaired t-test was used to estimate 83
the p values: *p<0.05. B, Mitochondria were isolated from 2-week-old WT, ftsh4-1 84
and ftsh4-2 seedlings grown under LD, 22°C and incubated at 22°C for 16 hours in 85
the presence of 5 mM succinate and 8 μM antimycin A, with or without of 3.5 mM 86
ATP. For protease inhibitor assay, the inhibitors of serine proteases (2 mM AEBSF) 87
and metalloproteases (25 mM ortho-phenanthroline, O-Phe) have been added into 88
the incubation medium. Mean values ± SD from three experiments are shown. The 89
unpaired t-test was used to estimate the p values: *p<0.05. 90
91
Figure 8. Mitochondrial morphology and class of lipids involved in cardiolipin 92
biosynthesis, differing in ftsh4. A, Mitochondrial morphology was observed by 93
scanning single protoplasts expressing GFP targeted to mitochondria of WT and 94
ftsh4-1 using Zeiss Confocal Microscopy LSM 510 Meta. Giant mitochondria are 95
highlighted with white arrows. Scale bar 5 µm. * An asteriks indicates a spherical 96
mitochondrion displaying reduced GFP fluorescence pointing out an occurence of 97
oxidative stress. B, Lipids of WT, ftsh4-1 and ftsh4-2 grown under optimal (22°C) and 98
moderately elevated temperature (30°C) were measured by mass spectrometry. Lipid 99
classes are shown in mol% of total lipids in the sample and are sums of individually 100
quantified lipid species. The unpaired t-test was used to calculate the p values: 101
p<0.05. Error bars correspond to SD (n=3). CL, cardiolipin; DAG, diacylglycerol; PC, 102
phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglicerol; PI, 103
phosphatidylinositol; PA, phosphatidic acid. Only selected classes are shown - see 104
Supplemental Fig. S4 for the complete data set. 105
106
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40
Figure 9. A hypothetical scenario of the events leading to the accumulation of 107
carbonylated proteins in mitochondria of A. thaliana in the absence of FTSH4. The 108
direction of short green arrows specifies change in abundance of cardiolipin (CL), 109
complex I (CI), complex V (CV), ROS, ATP and carbonylated proteins, in the 110
absence of FTSH4. Black lines ending with arrowheads indicate activating effects, 111
while perpendicular lines indicate inhibiting effects. The decreased content of CL as 112
well as absence of the chaperone-like activity of FTSH4 lead to lower stability/activity 113
of complex I and V, which in turn results in accumulation of ROS and decrease of 114
ATP, respectively. The lower concentration of ATP restricts activity of mitochondrial 115
ATP-dependent proteases, which are not able to degrade all carbonylated proteins 116
accumulating in consequence of the elevated ROS level. The lower content of CL 117
further restricts fission, which in turn causes the appearance of giant mitochondria 118
and also blocks mitophagy, important to eliminate mitochondria damaged by 119
oxidative stress. 120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
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41
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0
2
4
6
8
10
12
14
16
17 22 35 42
Ros
ette
dia
met
er (c
m)
Day after sowing (days) WT ftsh4-1 ftsh4-2
0
1
2
3
4
5
6
1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09
Del
ay in
leaf
dev
elop
men
t vs
WT
(day
s)
Growth stage ftsh4-1 ftsh4-2
Figure 1. Morphology of ftsh4 and WT plants growing under LD, 30°C. A, 2-week-old WT and ftsh4 plants grown on
agar plates. B, Delay time (in days) in leaf emergence of ftsh4-1 and ftsh4-2 compared to WT. Plants were staged as
described by Boyes et al. (2001). C, Rosette diameter of plants grown in soil at indicated time points after sowing.
Mean values ± SD from three measurements are shown. Significant differences are indicated by asterisks (one-sample
t-test *p<0.05). D, 5-week-old rosette leaves of plants grown in soil.
A
C
WT ftsh4-1 ftsh4-2
B
D
WT ftsh4-1 ftsh4-2
1 cm
WT ftsh4-1 ftsh4-2
1 cm * * * * * *
* *
* *
* *
*
*
*
*
*
* *
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Figure 2. A, Morphology of 2-week-old WT, ftsh4-1 and
ftsh4-1-FTSH4 (revertant) seedlings grown under LD,
30°C on agar plates. B, Carbonylated proteins in a total
protein extract of the above genotypes. Immunodetection
with anti-DNP antibodies and quantif ication of
carbonylated proteins in total protein extract, separated by
one-dimensional gel electrophoresis. Anti-DNP signals
from entire lanes were quantified densitometrically. In
each experiment, the values for the relative carbonylated
protein amount were calculated as a percentage of the
value determined for the wild type plants (set to 100%).
Mean values ± SD from at least three independent
experiments are shown. Statistically significant differences
in abundance between WT, ftsh4-1 and ftsh4-1-FtsH4
plants are indicated by asterisks (one-sample t-test
*p<0.05).
A
B
WT ftsh4-1 – FTSH4 ftsh4-1
0
20
40
60
80
100
120
140
160
180
Rel
ativ
e ca
rbon
ylat
ed p
rote
ins
leve
l
(% o
f WT)
WT ftsh4-1 ftsh4-1-FTSH4
*
1 cm
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Figure 3. Comparison of mitochondrial carbonylated proteins from WT (left) and ftsh4-1 (right) plants growing in LD,
30°C. Proteins separated by IEF/SDS two-dimensional gel electrophoresis were transferred on PVDF membrane to
subsequently detect carbonylated proteins using the OxyBlot technique. Arrowheads refer to protein spots
accumulating in ftsh4 mitochondria, which are identified in all tested setups. Protein spots are listed in Table II and
Supplemental Table S2.
pH11$pH$11$94$
66$
45$
14$
20$
30$
pH$3$
MSD1$
SHM1$MPP$B$
ATP1$
Succ$Co$Ligase$
ATP2$
FUM1$
GDC@T$
MTLPD1$
94$
66$
45$
14$
20$
30$
pH$11$pH$3$
Succ$Co$Ligase$
FUM1$ATP1$
GDC@T$
MTLPD1$
MSD1$
SHM1$MPP$B$
ATP2$
MW$$(kDa)$
MW$$(kDa)$
pH$11$WT ftsh4-1
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Figure 4. Relative transcript levels for selected genes encoding mitochondrial proteins in ftsh4-1 and ftsh4-2 mutants growing in LD, 30°C
compared to wild type plants. A, The level of transcripts for genes encoding proteins identified by DIGE analysis. B, The level of
transcripts for genes encoding proteins usually up-regulated by oxidative stress. Relative abundance of transcripts is expressed as log2
ratios. Mean values ± SD from at least three independent experiments are shown. The dotted lines indicate the cut-off value +/-0.5 (log2)
of the ratio corresponding to the threshold level for significant up- and down-regulation of the transcripts in ftsh4. Full names of genes are
given in Table I and Supplemental Table S1.
A
B
WT
ftsh4-1 ftsh4-2
Photorespiration and carbon metabolism
Chaperones and stress related
TCA cycle adjacent
Amino acid metabolism Contam. C I C II ATP synthase TCA cycle Proteases Transport
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!"
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!"
WT
ftsh4-1 ftsh4-2
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!"
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!"
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Figure 5. A, Relative transcript levels for genes encoding mitochondrial ATP-dependent proteases in ftsh4-1 and ftsh4-2
mutants grown in LD, 22°C and LD, 30°C compared to wild type plants. The relative abundance of transcripts is expressed
as log2 ratios. Mean values ± SD from at least three independent experiments are shown. The dotted lines indicate the cut-
off value +/- 0.5 (log2) of the ratio corresponding to the threshold level for significant up- and down-regulation of the
transcripts in ftsh4. B, Representative images of immunodetection of selected mitochondrial proteases in ftsh4-1 mutant
compared to wild type plants growing in LD, 22°C, LD, 30°C or SD, 22°C. C. Densitometric quantification of immunoblots
presented in B. Intensity of bands was estimated using Image Quant software (Molecular Dynamics). Data for ftsh4-1 are
expressed as percentage of the value for wild type plants. Mean values ± SD from at least three experiments are shown.
Significant differences in abundance between WT and ftsh4-1 mutant are indicated by asterisks (one-sample t-test
*p<0.05).
A
B
C
0
20
40
60
80
100
120
140
160
180
LD, 22°C LD, 30°C SD, 22°C LD, 22°C LD, 30°C SD, 22°C
FTSH10 FTSH3
Rel
ativ
e pr
otei
n ab
unda
nce
(% o
f WT)
WT ftsh4-1
* *
*
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Figure 6. Amounts and activities of respiratory complexes (A,B,C) and the level of ATP (D) in ftsh4 and WT plants.
Mitochondria were isolated from 3-week-old WT and mutant plants (ftsh4-1 and ftsh4-2) growing hydroponically in LD,
22°C and LD, 30°C. A, B Coomassie brilliant blue (CBB) and in-gel activity staining of complex I (CI) and V (CV) after
blue-native polyacrylamide gel electrophoresis. C, Quantification of activities of complexes I and V. The intensity of
bands was estimated by densitometric analysis using Image Quant software (Molecular Dynamics). Relative complex
activity from mutant mitochondria was calculated as percentage of that in WT plants. Differences in activity between
WT and mutants are in all cases statistically significant (in one-sample t-test p<0.05). Mean values ± SD from three
experiments are shown. D, The ATP content in WT and ftsh4 mitochondria. Mitochondria were isolated from 3-week-
old WT, ftsh4-1 and ftsh4-2 seedlings grown under LD, 30°C. The ATP concentration was determined as described in
Materials and Methods. The unpaired t-test was used to estimate the p values: *p<0.05. Error bars correspond to SD
(n=6).
C I C V CBB
I+III2
I V
III2
C I C V CBB
WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2
A B LD, 30°C LD, 22°C
C
WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2
I+III2
I V
III2
0
20
40
60
80
100
120
LD 22°C LD 30°C LD 22°C LD 30°C
C I C V
Rel
ativ
e ac
tivity
(% o
f WT)
WT ftsh4-1 ftsh4-2
D
* *
0
1
2
3
4
5
6
7
8
9
WT ftsh4-1 ftsh4-2
* *
ATP
cont
ent (
pmol
/mg
prot
ein)
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A
LD, 30°C
0h 16h 16h
WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2
Time [h] ATP - - - - - - + + +
B LD, 22°C; antimycin A
- - - - - - + + + + + + + + +
WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2 WT ftsh4-1 ftsh4-2
Time [h]
ATP
0h 16h 16h 16h 16h
O-Phe AEBSF - - - - - - - - - + + + - - -
- - - - - - - - - - - - + + +
Figure 7. In vitro carbonylated protein degradation in the WT and ftsh4 mitochondria. Immunodetection of
carbonylated proteins separated by one-dimensional gel electrophoresis was estimated with anti-DNP antibodies.
Anti-DNP signals from entire lanes were quantified densitometrically. Mean values ± SD from three experiments are
shown. A, Mitochondria were isolated from 2-week-old WT, ftsh4-1 and ftsh4-2 seedlings grown under LD, 30°C and
incubated at 22°C for 16 hours in the absence or presence of 3.5 mM ATP. The unpaired t-test was used to estimate
the p values: *p<0.05. B, Mitochondria were isolated from 2-week-old WT, ftsh4-1 and ftsh4-2 seedlings grown under
LD, 22°C and incubated at 22°C for 16 hours in the presence of 5 mM succinate and 8 µM antimycin A, with or
without of 3.5 mM ATP. For protease inhibitor assay, the inhibitors of serine proteases (2 mM AEBSF) and
metalloproteases (25 mM ortho-phenanthroline, O-Phe) have been added into the incubation medium. Mean values ±
SD from three experiments are shown. The unpaired t-test was used to estimate the p values: *p<0.05.
0
20
40
60
80
100
120
140
160
Rel
ativ
e le
vel o
f car
bony
late
d pr
otei
ns
(% o
f the
leve
l in
time
0h)
0h 16h - ATP 16h + ATP
WT ftsh4-1 ftsh4-2
* * *
0
20
40
60
80
100
120
140
160
180
Rel
ativ
e le
vel o
f car
bony
late
d pr
otei
ns
(% o
f the
leve
l in
time
0h)
0h 16h-ATP 16h+ATP 16h+ATP+AEBSF 16h+ATP+O-Phe WT ftsh4-1 ftsh4-2
*
* *
*
*
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Figure 8. Mitochondrial morphology and class of lipids involved in cardiolipin biosynthesis, differing in ftsh4. A,
Mitochondrial morphology was observed by scanning single protoplasts expressing GFP targeted to mitochondria of
WT and ftsh4-1 using Zeiss Confocal Microscopy LSM 510 Meta. Giant mitochondria are highlighted with white
arrows. Scale bar 5 µm. * An asteriks indicates a spherical mitochondrion displaying reduced GFP fluorescence
pointing out an occurence of oxidative stress. B, Lipids of WT, ftsh4-1 and ftsh4-2 grown under optimal (22°C) and
moderately elevated temperature (30°C) were measured by mass spectrometry. Lipid classes are shown in mol% of
total lipids in the sample and are sums of individually quantified lipid species. The unpaired t-test was used to
calculate the p values: p<0.05. Error bars correspond to SD (n=3). CL, cardiolipin; DAG, diacylglycerol; PC,
phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglicerol; PI, phosphatidylinositol; PA,
phosphatidic acid. Only selected classes are shown - see Supplemental Fig. S4 for the complete data set.
B
A
LD, 30° C
LD, 22° C
WT-GFP ftsh4-1-GFP
22° 30° 22° 30° 22° 30° 22° 30° 22° 30° 22° 30° 22° 30° 0
5
10
15
20
25
30
35
40
45
50
mol
%
Lipid class
* * * * *
CL DAG PC PE PG PI PA
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Figure 9. Figure 9. A hypothetical scenario of the events leading to the accumulation of carbonylated proteins in mitochondria of A.
thaliana in the absence of FTSH4. The direction of short green arrows specifies change in the abundance of cardiolipin (CL),
complex I (CI), complex V (CV), ROS, ATP and carbonylated proteins, in the absence of FTSH4. Black lines ending with
arrowheads indicate activating effects, while perpendicular lines indicate inhibiting effects. The decreased content of CL and a lack
of chaperone-like activity of FTSH4 lead to a reduced stability/activity of complex I and V, which in turn results in higher amount of
ROS and decrease of ATP. The lower concentration of ATP restricts activity of mitochondrial ATP-dependent proteases, which are
not able to degrade all carbonylated proteins accumulating in consequence of the elevated ROS level. The lower content of CL
further restricts fission, which in turn causes the appearance of giant mitochondria as well as blocks mitophagy, important to
eliminate mitochondria damaged by oxidative stress. OMM-mitochondrial outer membrane; IMS-intermembrane space of
mitochondria; IMM-inner mitochondrial membrane; FTSH- filament-forming temperature- sensitive, ATP-dependent
metalloprotease of mitochondrial inner membrane; LON-ATP-dependent serine protease of mitochondrial matrix; Clp- ATP-
dependent serine protease of mitochondrial matrix; PHB complex-prohibitin complex; CI-V-oxidative phosphorylation complex I-V;
CL-cardiolipin; ROS-reactive oxygen species; ATP- adenosine triphosphate.
FTSH4
CL#
CL#
ROS#
CARBONYLATED PROTEINS
ATP#
IMS
IMM
OMM
MATRIX
CIV$
FISSION MITOPHAGY
GIANT MITOCHONDRIA #
FTSH3$ FTSH10$ FTSH3/10$
PHB$$complex$
PHB$$complex$
PHB$$complex$
LON1$
CLPX(1;3)$LON4$
FTSH11$
CI$
CIII$
CII$
CV$
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