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Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables 1
Characterization of Mutations Associated with Retinal Disease 2
Benjamin M. Scott,* Steven K. Chen,* Nihar Bhattacharyya,* Abdiwahab Y. Moalim,* Sergey 3
V. Plotnikov,* Elise Heon,† Sergio G. Peisajovich,* Belinda S.W. Chang *,‡,§ 4
5
*Department of Cell and Systems Biology, University of Toronto, ON, Canada 6
†Department of Ophthalmology, Hospital for Sick Children, Toronto, ON, Canada 7
‡Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, ON, Canada 8
§Centre for the Analysis of Genome Evolution and Function, University of Toronto, ON, Canada 9
Author for correspondence: Belinda S.W. Chang belinda.chang@utoronto.ca 10
Keywords: Visual degenerative disease; retinitis pigmentosa; G protein-coupled receptor; disease 11
model; rhodopsin 12
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Abstract 14
G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in 15
eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number 16
of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but 17
can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult 18
for the visual pigment rhodopsin, a GPCR activated by light, for which hundreds of mutations 19
have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa (RP). In 20
this study, we successfully engineered, for the first time, activation by human rhodopsin of the 21
yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel 22
assay for rhodopsin light-dependent activation with studies of subcellular localization, and the 23
Genetics: Early Online, published on December 4, 2018 as 10.1534/genetics.118.301733
Copyright 2018.
upregulation of the unfolded protein response (UPR) in response to misfolded rhodopsin protein. 24
We use these assays to characterize a panel of rhodopsin mutations with known molecular 25
phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some 26
interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes 27
from patients with novel disease-linked mutations. We demonstrate that our engineered yeast 28
strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular 29
mechanisms underlying their pathogenicity. This approach may also be applied to better 30
understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as 31
a tool for investigating molecular mechanisms relevant to human disease. 32
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Introduction 34
The diversity of biologically relevant signals that G protein-coupled receptors (GPCRs) 35
detect make them critical to how cells sense and respond to their environment. Missense 36
mutations within this large family of cell surface receptors can therefore have serious 37
physiological effects, and many human diseases have been linked with missense mutations in 38
GPCRs (Heng et al. 2013). Over 63,000 missense mutations have been identified in human 39
GPCRs via large scale human genome studies (Pandy-Szekeres et al. 2018), but the majority 40
have not been studied in detail, so the significance to human health remains unclear. Missense 41
mutations can disrupt GPCR function in many ways, ranging from ligand binding, to protein 42
stability, to changes in downstream signaling and interactions with negative regulators (Stoy and 43
Gurevich 2015). Consequently, disease can arise through many different molecular phenotypes 44
and not all mutations are disease causing. 45
To better understand how human missense mutations contribute to disease, the yeast S. 46
cerevisiae has emerged as a powerful tool for characterizing human protein function due to 47
conserved molecular pathways, rapid growth, and ease of genetic manipulation (Laurent et al. 48
2016). These benefits have facilitated yeast models of human disease (Outeiro and Lindquist 49
2003, Perocchi et al. 2008), and studies of pathogenic human mutations (Sun et al. 2016, Yang et 50
al. 2017). Synthetic biology approaches, where human protein function and interactions are 51
quantified by yeast-based gene circuits, have enabled high-throughput experiments at an 52
impressive scale (Starita et al. 2015, Woodsmith et al. 2017, Weile et al. 2017, Sokolina et al. 53
2017). With genetic engineering, human GPCRs can be functionally linked to the yeast mating 54
pathway, and mating-responsive reporter genes have allowed for detailed studies of GPCR 55
activation (Liu et al. 2016). As human GPCRs retain their natural preferences for ligands and G 56
proteins in yeast (Brown et al. 2000), this application of synthetic biology combines the high-57
throughput capabilities of yeast-based studies with the ability to rapidly characterize GPCR 58
function in a cellular context. This has facilitated screens of chemical libraries for novel GPCR 59
ligands (Campbell et al. 1999, Horswill et al. 2007), and screens of mutated GPCRs to 60
characterize specific protein domains or to engineer novel function (Armbruster et al. 2007, 61
Erlenbach et al. 2001, Liu et al. 2015). 62
Despite the power of yeast-based studies of human GPCRs, only a small proportion of 63
GPCRs have been functionally linked to the yeast mating pathway, and all have been ligand-64
activated (Liu et al. 2016). Unlike these GPCRs, rhodopsin exists as a covalent complex between 65
its light-sensitive chromophore 11-cis retinal and the seven-helix transmembrane opsin 66
apoprotein (Smith 2010). Light exposure isomerizes the chromophore, which induces a 67
conformational change in rhodopsin’s transmembrane helices, activating the associated 68
heterotrimeric G protein, transducin, and triggering the visual transduction cascade that 69
eventually results in a signal to the brain that light has been perceived (Smith 2010). Not 70
surprisingly, missense mutations in rhodopsin are often associated with retinal diseases in 71
humans (Athanasiou et al. 2018). Retinitis pigmentosa (RP) is a highly heterogeneous, 72
degenerative retinal disorder that results in vision impairment and in some cases eventually 73
blindness, affecting approximately 1 in 4000 people worldwide (Fahim et al. 2000). The 74
heterogeneous nature of this degenerative disease has contributed to the difficulties in developing 75
effective prognoses and treatment. Missense mutations in rhodopsin have been associated with 76
20-30% of autosomal dominant RP and 1% of autosomal recessive RP cases, making rhodopsin 77
one of the most important RP genes (Fahim et al. 2000). Over 150 rhodopsin missense mutations 78
have been associated with disease (Stenson et al. 2014), and an additional >200 uncharacterized 79
mutations in rhodopsin exons are listed in the Genome Aggregation Database (Lek et al. 2016). 80
Without a tool to rapidly assess rhodopsin function, this increasing availability of genetic 81
information has yet to lead to a better understanding of pathogenicity of mutations associated 82
with RP and other inherited visual diseases. Determining the impact of missense mutations on 83
the ability of rhodopsin to respond to light currently relies on in vitro biochemical assays that are 84
labor intensive, requiring mammalian cell culture to produce one mutant protein at a time, 85
followed by immunofluorescence or immunoaffinity purification (Reeves et al. 1996, Sung et al. 86
1991). These technical challenges are compounded by the diverse molecular phenotypes of 87
rhodopsin mutations, ranging from constitutively active, to improper subcellular localization, to 88
disrupted post-translational modifications (Athanasiou et al. 2018). To efficiently characterize 89
the wide variety of patient-derived mutations, a rapid method that reliably recapitulates light-90
dependent signaling of rhodopsin is needed. 91
Here, we use synthetic biology approaches to engineer rhodopsin coupling to the yeast 92
mating pathway, demonstrating for the first time successful rhodopsin light-activated signal 93
transduction that can be rapidly quantified using a fluorescent reporter gene of mating pathway 94
activation. We compared our novel yeast-based assay to more established mammalian cell-based 95
methods, using a panel of previously-studied rhodopsin mutations. We found that measurements 96
of rhodopsin activation in yeast resemble in vitro results using rhodopsin purified from 97
mammalian cells, with some exceptions. We also found that a yeast-based reporter of the 98
unfolded protein response (UPR) produced results consistent with previous studies in 99
mammalian cells quantifying the effects of rhodopsin pathogenic mutants on cellular stress. 100
Finally, we used our combined approaches in yeast to investigate recently identified rhodopsin 101
mutations in patients with retinal disease, and were able to propose pathogenic classifications 102
that are supported by mammalian cell and clinical data. 103
Materials and Methods 104
Yeast Strain Engineering. The parent yeast strain for all strain engineering was CB008, 105
genotype W303 MATa, far1Δ, his3, trp1, leu2, ura3 (Table S1 contains all strain genotypes). All 106
gene knock-outs and knock-ins were conducted using homologous recombination of selectable 107
markers. pFUS1-mCherry was integrated at the MFA2 locus using plasmid pJW609 containing 108
the KanR marker. pFUS1 was defined as the 1636 bp immediately upstream of the Fus1 start 109
codon, the mCherry sequence used is from (Keppler-Ross et al. 2008), and ~1 kb homology 110
regions were used. STE2 and SST2 were targeted for deletion using TRP1 and HygB selectable 111
markers respectively, each with 180 bp of flanking homology regions identical to the sequences 112
flanking the ORF. The 5 C-terminal amino acids of Gpa1 (KIGII) was replaced with a Gpa1-Gαt 113
(transducin) chimera, containing the C-terminal amino acids from mammalian Gαt (DCGLF), 114
using plasmid pBS600 designed for this study (Figure S1), containing selectable marker LEU2, 115
and a sequence homologous to the 800 bp 3’ to natural Gpa1 gene. The C. albicans Adh 116
terminator was used downstream of both the pFUS1-mCherry and Gpa1-Gαt gene cassettes. 117
Strains were confirmed by PCR and flow cytometry. 118
Rhodopsin Mutation Selection and Patient Phenotyping. Rhodopsin mutations were selected 119
from across phenotypic classes, as reported in a recent comprehensive review (Athanasiou et al. 120
2018), and with at least one of the following assays previously published: transducin activation, 121
localization in mammalian cells, or spectroscopy indicating chromophore binding. The patient 122
cases were selected from an internal database and the phenotype information was collected 123
retrospectively. Other than basic demographic and genetic information, we collected information 124
about visual acuity (VA), color vision, Goldmann visual fields (GVF), electroretinography 125
(ERG) and imaging. Imaging included fundus photography (VisucamNM/FA - Carl Zeiss Meditec, 126
Dublin, California, USA and Optos), Optical coherence tomography (OCT, Cirrus from Carl 127
Zeiss Meditec, Dublin, California, USA). Genetic testing was done using gene panels based 128
sequencing by CLIA approved laboratories. This study was approved by the Human Research 129
Ethics Board of the hospital for Sick Children and met the Tenets of the declaration of Helsinki. 130
Cloning and Mutagenesis. The human rhodopsin sequence (RefSeq NP_000530.1) was 131
amplified using Pfu polymerase (Thermo) from plasmid pJET HuRh (Morrow et al. 2017), using 132
primers to insert flanking AarI restriction sites. Following AarI digestion, the rhodopsin 133
sequence was ligated to the yeast centromere plasmids pRS316 and pRS313 which each 134
contained the TDH3 promoter (pTDH3; alternatively called pGPD). For mammalian expression, 135
rhodopsin mutations were introduced into the wild-type bovine rhodopsin sequence in the p1D4 136
vector for immunoaffinity purification or the pGFP vector for SK-N-SH immunofluorescence 137
microscopy (Figure S2). Mutagenesis was conducted via PCR following the QuikChange site-138
directed mutagenesis protocol (Agilent) and using PfuUltra II Fusion HS DNA Polymerase 139
(Agilent). Mutagenesis primers were designed with 20 or 21 nucleotides identical to human or 140
bovine rhodopsin, flanking the mutant nucleotide(s). 141
Yeast Plasmid Transformation. Yeast strain BS017 or yJW1200 were transformed with 142
individual plasmids by standard lithium-acetate method and plated on selective media (SD-URA 143
or SD-HIS respectively). 144
Rhodopsin Purification. Culturing and transfection of HEK293T cells was performed as 145
previously described (Bhattacharyya et al. 2017). Briefly, p1D4 vector containing a rhodopsin 146
gene was transfected with Lipofectamine 2000 (Invitrogen) and cells were harvested after 48 147
hours. Rhodopsin was regenerated with 11-cis retinal for 2 hours before solubilization in 1% 148
dodecylmaltoside (Anatrace) and immunoaffinity purified with 1D4 monoclonal antibody 149
(Molday and MacKenzie 1983). The ultraviolet-visible absorption spectra of the rhodopsin 150
proteins were recorded using a Cary 4000 double beam spectrophotometer (Agilent). Pigments 151
were light bleached with a Fiber-Lite MI-150 high intensity illuminator (Dolan-Jenner) for 60 152
seconds at 20ºC. Dark-light difference spectra were calculated by subtracting the light-bleached 153
absorbance spectra from the dark spectra. 154
Yeast Light Activation Assay. Yeast strain BS017 transformed with a human rhodopsin mutant 155
gene in the pRS316 pTDH3 vector was incubated overnight in SD-URA media in a 30oC shaker. 156
The same strain transformed with a plasmid not containing the rhodopsin sequence (Vector) was 157
used as a negative control. Cells were diluted to OD600 0.05 in fresh media containing 5 μM 9-cis 158
retinal (Sigma-Aldrich) and incubated for 2 hours protected from light in a 30°C shaker. 9-cis 159
retinal is a common alternative to the natural chromophore 11-cis retinal, and gives comparable 160
in vitro results (Opefi et al. 2013). LightSafe 50 mL centrifuge tubes (Sigma-Aldrich) were used 161
for 5 mL cultures, and 96-well deep well blocks (VWR) wrapped in aluminum foil for 600 μL 162
cultures. Indicated cultures were then exposed to light using a Fiber-Lite MI-150 high intensity 163
illuminator (Dolan-Jenner) set to full intensity for 15 minutes at room temperature. After 100 μL 164
samples were taken for analysis, an additional 5 μM 9-cis retinal was added to indicated culture, 165
both light exposed and cultures kept in the dark, and placed back in the 30oC shaker. Light 166
exposure followed by retinal addition was conducted every hour for a total of six hours following 167
the first light exposure. Cells were then treated with the protein synthesis inhibitor 168
cycloheximide, to a final concentration of 10 μg/mL. The mCherry signal of at least 6,000 cells 169
was measured for each sample with a Miltenyi Biotec MACSQuant VYB. The mean mCherry 170
fluorescence was determined using FlowJo. After subtracting the mCherry fluorescence signal of 171
the Vector control, fluorescence values were normalized to the wild-type rhodopsin control used 172
in the same experiment, to allow comparisons between experiments performed on different days. 173
Immunofluorescence Microscopy. SK-N-SH neuroblastoma (ATCC HTB-11) cells were 174
grown and cultured in full media (DMEM (Life technologies), 10% FBS (Invitrogen), and 175
Penicillin-Streptomycin (Invitrogen)) at 37º in 5% CO2 and seeded into 24-well plates with 176
coverslips (Sarstedt) while under 5 passages. Once cells reached approximately 75% confluence, 177
they were transfected with 645 ng of pGFP plasmid containing the appropriate bovine rhodopsin 178
gene, using Lipofectamine 2000 (Invitrogen) protocols. After 24 hours, half the wells were 179
incubated with WGA in HBSS for 10 minutes at 37º to label the plasma membrane. All cells 180
were then rinsed with PBS and fixed with 2% paraformaldehyde in PBS. To label cells with the 181
endoplasmic reticulum marker antibody anti-calreticulin (1:400, Abcam), cells were washed and 182
permeabilized in PBS containing 1% bovine serum albumin (Sigma) and 0.1% saponin (PBS-183
BS). Anti-calreticulin was diluted in PBS-BS and incubated for 1hr at room temp. After washing 184
with PBS-BS, secondary antibody (Cy3-conjugated goat anti-rabbit IgGt, 1:200, Jackson 185
Immunoresearch) was diluted in PBS-BS and added to the wells for 1 hour. Nuclei were stained 186
with Hoechst (1:1000 in PBS, Hoechst type 33258 Invitrogen) for 10 minutes. Cells were 187
mounted with ProlLong Gold Antifade (Thermofisher), a coverslip as applied, and allowed to 188
cure for 24 hours in the dark prior to imaging on Leica TCSSP8 confocal microscope. ImageJ 189
was used to construct Z-stacks, maximum projection images and scale bars. 190
Yeast Microscopy. Yeast strain BS017 expressing human rhodopsin C-terminally tagged with 191
GFP were grown to log phase in selective media, then plated on glass-bottomed dishes (Greiner 192
Bio-one) treated with 1 mg/mL concanavalin A (Sigma-Aldrich). The centromere plasmid 193
pRS316 pTDH3 was used, and the GFP sequence used is from (Moser et al. 2013). Restriction 194
enzyme sites introduced a short amino acid linker (GGERGS) between the final rhodopsin 195
residue and first GFP residue. Images of the cells adhered to the dishes were acquired using a 196
Leica TCS SP8 confocal microscope with a 100X 1.4NA objective and a hybrid detector (Leica). 197
GFP fluorescence was analyzed using a custom Fiji-MATLAB pipeline (File S1, File S2), which 198
was similar to analyses performed to quantify fluorescence peaks in mammalian cells (Cheng et 199
al. 1999). On Fiji (Schindelin et al. 2012), yeast cell maps were generated by first removing 200
high-frequency noise using ROF Denoise (theta=25) and preparing for thresholding using 201
Enhance Contrast (0.3 saturation with Normalization). Manual thresholding, filling holes, and 202
finally selecting of cells using the watershed plugin generated final cell maps. Cell membrane 203
maps were defined in MATLAB as the 7 outer pixels (~0.7 µm) of each cell map. Fluorescent 204
patches at the cell membrane were defined in MATLAB as pixels with fluorescence intensity at 205
least 2.0 standard deviations above the edge mean. Cells with patchy membrane expression of 206
rhodopsin were defined in MATLAB as cells with at least 10% of their cell membrane 207
containing membrane patches. Bootstrapped standard errors were generated in MATLAB by 208
using the Statistics and Machine Learning Toolbox functions (Mathworks). 209
UPR Activation Assay. Yeast strain yJW1200 was generously provided by the Weissman lab, 210
University of California, San Francisco. This strain contains a 4x repeat of the unfolded protein 211
response element upstream of GFP, and the constitutive TEF2 promoter upstream of RFP 212
(Jonikas et al. 2009). yJW1200 transformed with a human rhodopsin mutant gene in the pRS313 213
pTDH3 vector was incubated overnight in 3 mL SD-HIS media in a 30oC shaker. The same 214
strain transformed with a plasmid not containing the rhodopsin sequence (Vector) was used as a 215
negative control. Cells were diluted to OD600 0.2 in 600 μL fresh media and incubated for 4 216
hours in a 30°C shaker. Cells were then treated with the protein synthesis inhibitor 217
cycloheximide, to a final concentration of 10 μg/mL. The GFP and RFP signal of at least 10,000 218
cells was measured for each sample with a Miltenyi Biotec MACSQuant VYB. The mean GFP 219
and RFP fluorescence was determined using FlowJo. Fluorescence values were normalized to the 220
wild-type rhodopsin control used in the same experiment, to allow comparisons between 221
experiments performed on different days. 222
Statistical Analyses and Graphs. Statistical analyses were performed using Prism (GraphPad), 223
using one-way ANOVA for UPR comparisons to wild-type rhodopsin, and for yeast light and 224
dark activation comparisons to wild-type rhodopsin. Student’s t-test was used to compare the 225
results of one mutant or condition to one other mutant or condition. Graphs were generated using 226
Prism or Excel (Microsoft). 227
Data Availability Statement. Strains and plasmids are available upon request. All 228
supplementary figures, tables, and files have been uploaded to figshare. 229
230
Results 231
Human Rhodopsin Functionally Couples to the Yeast Mating Pathway and Signal 232
Transduction is Dependent on Light 233
We have engineered, for the first time, a vertebrate rhodopsin that can successfully 234
couple to the yeast mating pathway. This was accomplished by first knocking out the genes 235
encoding Far1, to prevent cell cycle arrest, and the GTPase-activating protein Sst2, a negative 236
regulator of the mating pathway (Brown et al. 2000). The endogenous mating pathway GPCR 237
Ste2 was also knocked out, to prevent unnecessary interactions with downstream mating 238
pathway proteins. Next, a chimeric G alpha protein and the fluorescent protein mCherry under 239
the regulation of the mating-responsive FUS1 promoter (pFUS1) were inserted into the yeast 240
genome (Figure 1, Table S1). As rhodopsin is known to only interact with G alpha proteins 241
containing the same five amino acid C-terminal sequence (transducin in rod photoreceptors and 242
Gαi1 in engineered systems (Sun et al. 2015, Maeda et al. 2014)), only one Gpa1 chimera (Gpa1-243
Gαt) was required for our study. To ensure functional coupling between Gpa1-Gαt and human 244
rhodopsin, we first expressed a known constitutively active rhodopsin mutant, E113Q M257Y 245
(Han et al. 1998). Consistent and high levels of mCherry fluorescence were observed, regardless 246
of the presence of retinal chromophore (Figure S3), indicative of productive rhodopsin 247
expression and the ability to activate the yeast mating pathway. 248
Wild-type human rhodopsin was then expressed using the same strain, and when 249
incubated with retinal, induced the expression of mCherry only in response to light (Figure 2). 5 250
μM 9-cis retinal in culture media was sufficient to elicit this light-dependent response, a 251
concentration also used for the heterologous expression of rhodopsin using mammalian cells 252
(Opefi et al. 2013). The 5- to 10-fold increase in mCherry fluorescence in response to GPCR 253
activation was comparable to previous reported activation of the natural mating pathway GPCR 254
Ste2 (Ishii et al. 2008, Kompella et al. 2017). Increasing the retinal concentration did not 255
increase activation of the mating pathway, indicating rhodopsin molecules were saturated with 256
chromophore (Figure S4). However, mating pathway response was enhanced when retinal was 257
added after each hourly light exposure, to account for the lack of retinal recycling enzymes in 258
yeast. 259
260
Magnitude of Light-Activated Signal Transduction in Yeast Comparable to Assays of 261
Rhodopsin Expressed in Mammalian Cells 262
After establishing light-dependent activation of rhodopsin in yeast, we next sought to 263
compare these new yeast-based methods to traditional in vitro methods utilizing protein purified 264
from mammalian cells. Previously characterized rhodopsin mutations P23H, M39R, and G51A 265
were specifically chosen to establish a gradient of phenotypic severity. P23H is the most 266
common RP-associated rhodopsin mutation in North America (Dryja et al. 1990, Mendes et al. 267
2005), and has been characterized in a number of cell and animal models. P23H rhodopsin 268
consistently displays poor stability (Krebs et al. 2010, Chen et al. 2014), aggregation in the ER 269
(Chiang et al. 2012b), and disrupted transducin activation (Opefi et al. 2013, Chen et al. 2014), 270
leading to severe retinal degeneration (Athanasiou et al. 2017, LaVail et al. 2018, Cideciyan et 271
al. 1998). The less severe M39R mutation, which is also associated with RP, has been studied 272
using both bovine and human rhodopsin genes displaying a more severe cytosolic aggregation 273
phenotype in the human gene background (Ramon et al. 2014, Davies et al. 2012). As M39R 274
rhodopsin is more productively expressed by mammalian cells than P23H rhodopsin, and a 275
proportion remains able to form a light-responsive complex with retinal, it was selected as an 276
intermediate RP-associated rhodopsin mutation (Davies et al. 2012, Ramon et al. 2014). G51A is 277
the most common nonsynonymous rhodopsin mutation in humans (Lek et al. 2016), displays a 278
less severe phenotype in vitro and in patients (Bosch et al. 2003, Cideciyan et al. 1998), and may 279
be an asymptomatic variant (Athanasiou et al. 2018). These three rhodopsin mutants were each 280
expressed using the mCherry reporter yeast strain. Following exposure to light, the relative 281
mCherry fluorescence was observed as follows: P23H < M39R < G51A = WT (Figure 3A). 282
Next, we compared rhodopsin activation in yeast to traditional in vitro methods for 283
determining rhodopsin function in response to light. The same three mutants were purified 284
following heterologous expression in mammalian cells, then regenerated with retinal. By 285
recording the absorption spectra before and after exposure to light, difference spectra showing 286
the response of rhodopsin to light could be measured. This method has been used extensively to 287
characterize missense mutations suspected to cause inherited retinal disease, as a measure of the 288
ability of rhodopsin to properly fold and respond to light (Sung et al. 1991, Opefi et al. 2013). 289
The relative response to light displayed a similar range of function as the yeast-expressed 290
mutants (Figure 3B). This suggested that not only is the function of yeast-expressed rhodopsin 291
similarly impacted by pathogenic mutations, but the relative activation of the mating pathway in 292
yeast is comparable to the severity of the mutant as measured in vitro using rhodopsin purified 293
from mammalian cells. 294
295
Pathogenic Mutations Known to Disrupt Rhodopsin Stability or G Protein Coupling 296
Prevent Light-Activated Signal Transduction in Yeast 297
After establishing yeast as a platform for quantifying light-dependent rhodopsin 298
activation, we investigated a larger panel of rhodopsin mutations to understand how the wide 299
range of known functional phenotypes translates to a response in yeast. Efforts have been made 300
to classify mutations based on these phenotypes, which range from completely inactive to 301
constitutively active (Figure S5, Table S2), as discussed in detail in a recent review (Athanasiou 302
et al. 2018). However, as this new yeast assay examines rhodopsin signaling in an engineered 303
cellular system, it was unknown how results would compare to traditional in vitro methods using 304
purified mammalian cell-expressed rhodopsin. We first focused on pathogenic mutations known 305
to disrupt rhodopsin folding and stability, as we hypothesized these loss-of-function mutations 306
would be easier to distinguish from wild-type. These mutations were placed into three groups 307
based on previous characterization: if the mutation intrinsically disrupts rhodopsin stability; if 308
the mutation indirectly affects stability by disrupting a post-translational modification 309
(glycosylation) motif; or if the mutation disrupts G protein coupling and leads to constitutive 310
endocytosis (Class 3). 311
When expressed in yeast and exposed to light, rhodopsin function was significantly 312
disrupted by each of the mutations known to result in misfolding or instability, with the 313
exception of D190N (Figure 4A). D190N had previously been shown to be a less severe RP-314
linked mutation (Tsui et al. 2008, Liu et al. 2013, Fishman et al. 1992), and ERG data from 315
patients matches our observation that this missense mutation does not completely disrupt 316
rhodopsin function (Sancho-Pelluz et al. 2012). The G89D and L125R mutations also had a 317
reduced but measurable response to light when expressed in yeast, which fits with previous 318
trends observed (Kaushal and Khorana 1994, Bosch et al. 2003). Interestingly, L125R lead to 319
signaling in the dark as well, equivalent to the mutant’s light-dependent activation, which has not 320
previously been reported. 321
Mutations in the N-terminal cap of rhodopsin (V20G, P23H, and Q28H) have been 322
functionally characterized in detail, and are known to poorly activate transducin in response to 323
light (Opefi et al. 2013). Similarly, mutations M39R, N55K, G106W, C110Y, G114D and 324
P171L have each been shown to disrupt or prevent productive formation of a opsin-retinal 325
complex (Davies et al. 2012, Ramon et al. 2014, Sung et al. 1991, Sung et al. 1993, Hwa et al. 326
1999, Andres et al. 2003), which matched our observation that light-activated signal transduction 327
was significantly impaired in yeast. 328
Mutations T4K, N15S, and T17M prevent glycosylation at residues N2 and N15, 329
resulting in a severe reduction in rhodopsin stability (Kaushal et al. 1994, Opefi et al. 2013). The 330
NXS/T glycosylation consensus sequence is recognized across eukaryotes (Lam et al. 2013), and 331
a previous study indicated that yeast-expressed bovine rhodopsin was glycosylated 332
(Mollaaghababa et al. 1996). The reduced stability of unglycosylated rhodopsin is known to 333
prevent productive transducin activation in vitro (Opefi et al. 2013), and comparable reductions 334
in signaling were observed in our yeast assay. A general trend of T4K (40% wild-type activity) 335
being less severe than N15S (4%) and T17M (15%) was observed, similar to previous studies 336
which indicated glycosylation is more important on N15 than it is on N2 (Kaushal et al. 1994, 337
Tam and Moritz 2009, Opefi et al. 2013). 338
Of the rhodopsin mutations we studied with impaired signaling, R135G is unique as it 339
does not cause misfolding and it does not prevent the formation of a stable complex with retinal, 340
when using rhodopsin purified from mammalian cells (Sung et al. 1993). R135G mutates the 341
highly conserved E/DRY motif, where R135 is the arginine residue in this motif and is crucial 342
for G protein coupling (Acharya and Karnik 1996, Rovati et al. 2007). In addition, when 343
heterologously expressed in mammalian cells, R135 mutations cause rhodopsin to be 344
hyperphosphorylated, leading to aggregation with visual arrestin and constitutively undergoing 345
endocytosis (Chuang et al. 2004). With two unique molecular mechanisms contributing to 346
pathogenicity, R135 mutants have been placed in their own “Class 3” category (Athanasiou et al. 347
2018, Chuang et al. 2004). The observed absence of signaling in yeast was in line with the 348
reported in vitro transducin activation defect using mammalian cell-derived R135G rhodopsin 349
(Min et al. 1993, Acharya and Karnik 1996). However, as our light-activated signal transduction 350
assay could not distinguish between disrupted G protein coupling versus aberrant endocytosis, 351
this assay alone could not determine the molecular mechanism behind the lack of R135G 352
signaling in yeast. 353
354
Pathogenic Mutations that Enhance or Do Not Disrupt Transducin Activation Respond 355
Similarly in Yeast 356
Based on the wild-type-like activity of G51A we observed in yeast, and the reduced or 357
inactive response of misfolded and unstable mutants, we hypothesized that rhodopsin mutations 358
which maintain or increase light-dependent activation in vitro may behave similarly in yeast. 359
Constitutively active rhodopsin mutants are also associated with disease, causing congenital 360
stationary night blindness (CSNB) and RP (Park 2014). We specifically selected a panel of 361
rhodopsin mutations to characterize in yeast where activity was known to vary greatly, from 362
asymptomatic, to constitutively active, to increased signaling in the dark. 363
Across this diversity of function, yeast-expressed rhodopsin again behaved comparably to 364
rhodopsin purified from mammalian cells, with both wild-type-like signals and increased 365
signaling observed depending on the mutation (Figure 4B). We grouped mutations known to 366
increase downstream transducin activation in vitro, although this increased activity can occur in 367
the light, dark, or in both states (Park 2014). The M44T mutation showed a significantly higher 368
response than wild-type, at over 1.6-fold wild-type, which matches in vitro transducin activation 369
data for M44T (Andres et al. 2003). T94I trended higher than wild-type, and is also believed to 370
cause CSNB due to constitutive activation (119% WT signaling in vitro) (Gross et al. 2003), but 371
the increase we observed versus wild-type (115% WT signaling in yeast) was not statistically 372
significant. V137M has been reported to activate transducin 1.25-fold greater than wild-type 373
(Andres et al. 2003), but we did not observe an increase in rhodopsin activity. The V137M 374
mutation is known to have highly variable clinical phenotypes (Ayuso et al. 1996), and it has 375
been suggested to be an asymptomatic variant (Rakoczy et al. 2011). 376
Of the rhodopsin mutations with known increased activity that we studied, S186W is 377
unique as it is believed to cause autosomal dominant RP due to increased signaling in the dark 378
(Liu et al. 2013). This is a result of reduced thermal stability of the inactive dark state, where 379
spontaneous thermal isomerization of the chromophore leads to signaling in the dark (Liu et al. 380
2013), a phenomenon called “dark noise” (Luo et al. 2011). The increased signaling that we 381
observed in the dark for S186W, equivalent to 30% of activated wild-type rhodopsin, fit with this 382
proposed mechanism of RP pathogenesis. E150K was also found to have significantly elevated 383
signaling in the dark, when expressed in yeast. This data is in line with a mouse model of 384
E150K, which was found to have elevated photoreceptor signaling in the dark, also believed to 385
be due to reduced thermal stability of the dark state (Zhang et al. 2013). The E150K mutation is 386
associated with autosomal recessive RP (arRP) and was previously shown to have a 1.3-fold 387
increased activation of transducin after light exposure (Zhang et al. 2013). An identical value 388
was observed using our yeast-based assay but was not statistically significant. 389
To determine if dark state signaling in yeast was retinal-dependent, we investigated 390
signaling without the addition of retinal for selected mutants (Figure S6). Elevated dark state 391
signaling appeared to be retinal-dependent for only M44T, L125R, E150K, and S186W. This fits 392
with the proposed mechanism of thermal isomerization of retinal contributing to dark noise for 393
the E150K and S186 mutants (Zhang et al. 2013, Liu et al. 2013), while providing new insight 394
on the M44T and L125R mutants. 395
Some rhodopsin mutations may cause disease by preventing the formation of rhodopsin 396
homodimers (Ploier et al. 2016), but their pathogenicity is debated due to their relatively high 397
frequency in sequenced human genomes (>1:80,000) (Athanasiou et al. 2018). The F45L and 398
V209M mutations were found to activate the mating pathway at wild-type levels when exposed 399
to light, matching published in vitro transducin activation assays (Ploier et al. 2016). F220L was 400
found to have a 1.5-fold greater response, which was unexpectedly higher than the wild-type-like 401
value reported for the F220C mutation (Ploier et al. 2016). Similarly, although V104I is 402
considered asymptomatic, light activation of the mating pathway was approximately 1.3-fold 403
greater than wild-type. This mutation does not segregate with RP in genetic studies (Macke et al. 404
1993), but transducin activation assays have not previously been performed, so it is unclear how 405
our results in yeast relate to a potential human phenotype. 406
Mutations in the C-terminus of rhodopsin disrupt trafficking to the rod outer segment 407
(ROS), but do not affect trafficking in other mammalian cell types and do not affect transducin 408
activation in vitro (Sung et al. 1994), therefore we did not expect their function to differ from 409
wild-type rhodopsin in yeast. Interestingly, V345M significantly affected light-activated 410
signaling in yeast, despite the mutation occurring in the C-terminus which is not believed to be 411
required for G protein activation. A study of transducin activation using V345M rhodopsin 412
purified from mammalian cells has not been performed to compare to our yeast results. 413
There were two examples where light-activated signaling in yeast differed from reported 414
in vitro results using rhodopsin purified from mammalian cells. The A292E mutation is 415
associated with CSNB, and has constitutive activation in vitro in the absence of retinal, but 416
reduced light-dependent transducin activation when retinal is supplied (Gross et al. 2003). In our 417
yeast signaling assay, a 2.1-fold increase in light-activated signaling versus wild-type was 418
observed, greater than any other mutation we studied. A292E signaling in the dark with retinal 419
added was not significantly increased, and was equivalent to not adding retinal. G90D, another 420
constitutively active mutant associated with CSNB (Rao et al. 1994), activated the mating 421
pathway at only 44% of wild-type’s response. This result was similar to one transducin 422
activation study using G90D (59% of wild-type) (Zvyaga et al. 1996), while others have shown 423
wild-type-like responses to light but constitutive activation in the absence of retinal, similar to 424
A292E (Gross et al. 2003, Rao et al. 1994). 425
426
Light-Activated Signal Transduction in Yeast Correlates with Published Assays of 427
Rhodopsin Function 428
Of the 33 mutations and controls studied, 23 had previously reported measurements of 429
light-dependent activation of transducin in vitro, or measurements of photoreceptor activity by 430
ERG (Table S2). When plotted together, our yeast-based measurements closely matched this 431
available data on rhodopsin signaling, approaching a 1:1 ratio (Figure S7). This held true for a 432
diverse array of phenotypes, ranging from pathogenic due to inactivation, or pathogenic due to 433
constitutive activation, to asymptomatic. A292E was found to be an outlier from this trend. The 434
rate of light-dependent transducin activation has been reported at ~80% of wild-type, although 435
this mutant has constitutive activation in the absence of retinal in vitro (Gross et al. 2003). As 436
signaling in yeast occurs in a cellular context, versus traditional in vitro assays that use purified 437
protein, the increase in light-activated signaling we observed for A292E may have been a 438
combination of signaling both with and without retinal bound, or may represent a unique 439
signaling state in yeast. Overall, however, the general trend strongly supports the use of yeast to 440
quantify rhodopsin mediated G protein signaling in response to light, as the yeast-based assay 441
was comparable to more laborious assays for characterizing patient derived mutations. 442
443
Subcellular Localization of Rhodopsin is Comparable Between Yeast and Mammalian 444
Cells 445
As the majority of disease linked rhodopsin mutations cause the receptor to misfold 446
(Athanasiou et al. 2018), comparing the subcellular localization of rhodopsin using mammalian 447
cells is a common technique to study protein trafficking and ER retention, and is predictive of 448
pathogenicity (Sung et al. 1991, Behnen et al. 2018). To establish phenotypes in mammalian 449
cells to compare to, we again used the rhodopsin mutations P23H, M39R, and G51A to create a 450
gradient of phenotypic severity. Rhodopsin mutants were expressed in SK-N-SH neuroblastoma 451
cells with a C-terminal GFP tag that has previously been shown to not affect rhodopsin stability 452
in vitro or in vivo (Moritz et al. 2001) 453
The P23H mutation has been characterized in a number of cell models, consistently 454
showing poor plasma membrane expression regardless of cell type (Sung et al. 1991, Chiang et 455
al. 2012b, Chiang et al. 2015). When expressed in SK-N-SH neuroblastoma cells, 456
immunohistochemistry revealed that P23H rhodopsin did not localize to the plasma membrane, 457
forming aggregates in the cytosol and colocalized with an ER-specific marker (Figure 5A, Figure 458
S8). M39R rhodopsin displayed a nearly wild-type phenotype, colocalizing with a plasma 459
membrane marker but with some evidence of mutant rhodopsin retained within the cell (Figure 460
S8), similar to a previous study (Davies et al. 2012). G51A displayed robust wild-type-like 461
localization on the plasma membrane, consistent with a recent report (Behnen et al. 2018). This 462
comparative range of subcellular localization matched what we previously observed in assays of 463
rhodopsin function for these three mutations. 464
We next sought to determine if the same rhodopsin mutants expressed in yeast trafficked 465
to the plasma membrane in a similar manner. The same yeast strain used to functionally couple 466
human rhodopsin to the mating pathway was used to express select rhodopsin mutants with GFP 467
fused to the C-terminus. Similar techniques have been used to investigate productive expression 468
of other human GPCRs in yeast, by observing expression on the plasma membrane or the 469
presence of aggregates (O'Malley et al. 2009). Mirroring our mammalian cell-based 470
observations, P23H and M39R were poorly distributed across the yeast plasma membrane, with 471
highly localized “patchy” expression, while G51A localized consistently to the membrane 472
(Figure 5A). The peri-nuclear ring observed is similar to the localization of other human GPCRs 473
expressed in yeast, indicating the ER membrane (O'Malley et al. 2009, Hashi et al. 2017). 474
Although P23H was observed to form aggregates in the ER of our mammalian cells, this was not 475
observed in yeast, however M39R did appear to form aggregates in the cytosol of yeast. 476
We expanded this microscopy-based analysis to additional rhodopsin mutants expressed 477
in yeast (Figure S9). Image analysis software was used to quantify rhodopsin distribution on the 478
plasma membrane of each yeast cell, identifying localized regions or “patches” where rhodopsin 479
appeared to aggregate (Figure 5B). Wild-type rhodopsin was observed to have a low number of 480
cells displaying a “patchy” phenotype, suggesting even distribution across the plasma membrane. 481
Some mutants associated with misfolding or reduced stability (i.e. N15S, M39R, L125R) 482
exhibited incomplete distribution on the plasma membrane, characterized by a “patchy” 483
phenotype. Mutations in the C-terminus of rhodopsin (Q344ter, V345M, P347L) disrupt the 484
VXPX motif, which is crucial for trafficking rhodopsin in photoreceptors (Wang and Deretic 485
2014). However, mutations within this motif do not affect rhodopsin localization in mammalian 486
cells that are not photoreceptors (Sung et al. 1991, Sung et al. 1993), and were not found to 487
affect rhodopsin localization in yeast. In general, these findings indicated that rhodopsin 488
maintains its subcellular localization when expressed in yeast, which is likely dependent on 489
rhodopsin folding and stability, just as it is with heterologous expression in mammalian cells. 490
491
The Yeast Unfolded Protein Response is Upregulated by Misfolded Rhodopsin Mutants 492
After discovering that the subcellular localization of rhodopsin and changes in responses 493
to light were preserved in yeast, we investigated additional molecular pathways known to be 494
affected by pathogenic rhodopsin mutations. Rhodopsin mutations P23H and T17M have been 495
shown to activate the UPR in mammalian systems, indicative of severe misfolding (Lin et al. 496
2007, Kunte et al. 2012). P23H has been shown to preferentially activate the IRE1 UPR pathway 497
in mammalian cells (Chiang et al. 2015), a pathway also present in yeast. Similar to mammalian 498
IRE1, yeast IRE1 serves as a sensor of misfolded protein in the ER, which activates the 499
transcription factor HAC1 (Kimata and Kohno 2011). Yeast strains and plasmids have been 500
devised utilizing a HAC1-responsive promoter to express reporter genes, which have been used 501
to predict productive expression of other human GPCRs in yeast, where greater UPR activation 502
was associated with GPCR misfolding and aggregation (O'Malley et al. 2009). Due to the 503
conserved pathway, and established results with other GPCRs, we hypothesized that the severity 504
of misfolded rhodopsin could be quantified using a yeast-based sensor of UPR upregulation. The 505
strain designed by Jonikas et al. (2009) has an additional gene cassette constitutively expressing 506
RFP, which can be used to correct for changes in global protein expression (Jonikas et al. 2009). 507
We used this strain to study the 33 selected rhodopsin mutations, plus controls, to quantify their 508
effect on UPR upregulation. 509
Expressed in the UPR-reporter strain, P23H and T17M recapitulated the expected 510
elevated UPR activation in yeast, in addition to several other mutations known to disrupt 511
rhodopsin stability (Figure 6). T4K and N15S, which like T17M disrupt glycosylation, also 512
upregulated the UPR, suggesting a crucial stabilizing nature of these posttranslational 513
modifications. C110Y prevents the formation of a critical disulphide bond in rhodopsin, severely 514
impacting stability and function (Hwa et al. 1999), and the observed increased UPR in yeast 515
matches in silico predictions that this mutant is highly unstable (Rakoczy et al. 2011). Elsewhere 516
in transmembrane helix three, the G114D and R135G mutations similarly increased the UPR. In 517
mammalian cells, R135G is hyperphosporylated and aggregates in endosomes (Chuang et al. 518
2004), but accumulation in the ER or activation of the mammalian UPR has not been reported. 519
That R135G activated the yeast UPR suggests that this mutant may be misfolded in yeast. 520
The constitutively active mutant A292E showed an upregulation in the UPR which has 521
not previously been reported, which may be due to the replacement of a small uncharged residue 522
with a large negatively charged residue proximal to the retinal binding pocket. A reduced UPR 523
for mutations M44T, E150K, and V345M compared to wild-type was observed, but the 524
physiological relevance is unclear. Interestingly, culturing in selective media alone was sufficient 525
to upregulate the UPR, revealed by the difference between the Vector control and the 526
untransformed strain grown in rich media. A log2 GFP/RFP ratio of ~1.0 was previously shown 527
to indicate moderate UPR upregulation (Jonikas et al. 2009), which was observed for the Vector 528
control prior to normalizing the data. Expressing wild-type rhodopsin gave a similar value, 529
which suggested baseline UPR upregulation was due to growth in selective media and not the 530
overexpression of rhodopsin. 531
532
Yeast Assays of Rhodopsin Mutations V81Δ, A164E, A164V Found in Retinal Disease 533
Patients 534
Having established a suite of new yeast-based techniques to investigate rhodopsin 535
functional and molecular phenotypes, we applied these approaches to study three rhodopsin 536
mutations found in degenerative retinal disease patients diagnosed with retinitis pigmentosa. One 537
of the rhodopsin mutations, V81Δ, is a new mutation that has not been previously reported (Case 538
1), and two of the other mutations are previously reported but have had little (A164V, Case 2), or 539
no experimental characterization with respect to those rhodopsin mutations (A164E, Case 3). 540
Missense mutations A164E and A164V have been previously linked to autosomal dominant RP 541
(Fuchs et al. 1994, Hwa et al. 1997), but the molecular mechanism underlying the disruption of 542
rhodopsin function at this site remains to be elucidated. Studies of A164V suggest that helical 543
packing may be an issue (Hwa et al. 1997, Stojanovic et al. 2003), but the effects of introducing 544
a charged residue at this site have not yet been investigated. Mutations at the same residue in 545
rhodopsin can have highly heterogenous phenotypes (Bosch et al. 2003), so we sought to 546
compare Al64E to A164V in greater detail. We identified a new V81Δ mutation in a patient 547
(Case 1) with early-onset autosomal dominant RP (Table S3). This mutation completely removes 548
the V81 codon from the rhodopsin DNA sequence, resulting in a deleted amino acid in the 549
central part of the second transmembrane domain (Figure 7A). Such an amino acid deletion 550
could disrupt alpha helix formation and stability in the membrane, and is likely to lead to a 551
severe molecular phenotype for V81Δ rhodopsin. 552
We characterized V81Δ, A164E, and A164V using the yeast-based methods for 553
investigating rhodopsin molecular phenotype and function, with experiments conducted in the 554
same manner as previously described (Figure 7B-E). Plasma membrane localization of all three 555
mutants were poor, with 28-40% of yeast cells displaying patchy expression of rhodopsin. Light 556
activated signal transduction in yeast was completely abolished for each, similar to the other 557
mutants we studied known to be unstable or misfold. UPR activation was elevated for all three 558
mutants, with A164E higher than any other rhodopsin mutant we studied. Together, our results 559
suggest that all three of these mutants have a severe phenotype in yeast, based on decreased 560
function, subcellular localization, and UPR activation, where we may expect a difference in 561
severity between the A164 mutants based on UPR activation. 562
563
Yeast Assays Comparable to Mammalian Cell Data for Rhodopsin Mutations V81Δ, 564
A164E, A164V 565
We compared our yeast-based assays of V81Δ, A164E, and A164V to expression in 566
mammalian cells. A164V colocalized with the plasma membrane marker, suggesting a less 567
severe phenotype in mammalian cells, which contrasted with the yeast data (Figure 8A). 568
However, A164E and V81Δ were completely retained inside mammalian cells, colocalizing with 569
the ER marker, indicating they were severely misfolded. Following immunoaffinity purification 570
from mammalian cells, the three RP-associated mutations also showed a range in their response 571
to light (Figure 8B). Heterologous expression of V81Δ produced no functional protein, 572
demonstrating a very severe phenotype. A164E, while not as severe phenotype as V81Δ, 573
expressed poorly and produced limited functional protein. A164V expression did produce a high 574
amount of functional protein, consistent with another in vitro study of this mutation (Stojanovic 575
et al. 2003). The sum of this cellular and functional data suggested the same functional trend we 576
first predicted using yeast-based methods, although there was more evidence of functional 577
A164V protein in mammalian cells. 578
579
Patient Clinical Data Supports Functional Trend Predicted by Yeast and Mammalian Cells 580
Next, we looked at patient clinical data. The comparative severity in vitro was found to 581
follow the same trend as available patient phenotype information (Table S3). The most severe 582
phenotype was exhibited by the patient with the V81Δ mutation (Case 1). This patient first had 583
symptoms around 10 years of age, with difficulty adapting to a dim lit environment (nyctalopia). 584
This slowly progressed, and at 39 years she has moderate visual acuity loss (20/50), mildly 585
abnormal color vision, constriction of the visual field to the central 5 degrees. At age 26 years 586
electroretinography already documented severe reduction of rod and cone function. The 587
phenotype of the patient with the A164V mutation (Case 2) is milder than that of the patient with 588
the A164E mutation. Although Case 2 had symptoms of nyctalopia since childhood, the 589
progression of his disease was extremely slow. At age 64 years his electroretinogram was 590
recordable and only mildly abnormal. His central visual acuity at 67 years was 20/40 and despite 591
a paracentral scotoma (area of decreased vision), he maintained a peripheral field. In contrast the 592
patient with the A164E mutation (Case 3) has a good central visual acuity and normal color 593
vision at age 53 years. However, her paracentral scotoma were more severe and progressed to 594
form an annular scotoma at the age of 53 years. Unlike Case 1 (V81Δ), she also preserved some 595
good peripheral field of vision at the age of 45 years, and her ERG was only moderately 596
abnormal. 597
The V81Δ patient is the youngest of the three with a highly reduced retinal function, 598
while the A164V patient is the eldest of the three with the best retinal function (Figure 9). A164E 599
again appeared intermediate to both. These results show that the overall trend of clinical severity 600
was accurately predicted by combining both sets of yeast-derived and mammalian cell-derived 601
data. The yeast methods provided additional information on UPR upregulation which also 602
supported the difference in severity between A164E and A164V mutations, while being less 603
labor intensive than mammalian microscopy and expression methods. 604
605
Discussion 606
Yeast Provide New Methods to Investigate Rhodopsin Structure and Function 607
In this study, we have engineered the yeast S. cerevisiae to characterize both known and 608
novel pathogenic mutations of the visual pigment rhodopsin. In comparing our new assays to 609
traditional mammalian cell-based approaches, we demonstrate that the molecular phenotypes of 610
this light-activated human GPCR are similar in yeast, and that these phenotypes reflect patient 611
clinical data. There are a number of advantages and differences when compared to traditional 612
techniques, as these yeast-based rhodopsin assays are performed in a cellular context, which 613
provides a new perspective on signal transduction pathways, subcellular localization, and UPR 614
upregulation. 615
A direct measurement of downstream pathway activation in response to light was 616
achieved by functionally coupling rhodopsin to the yeast mating pathway. This required 617
productive in vivo activation of a G protein, mimicking the initial step that occurs in human 618
photoreceptors, even if downstream signaling differs. By using yeast, many rhodopsin mutations 619
could be studied in the same experiment, with multiple replicates, without laborious purification 620
steps which are traditionally required for in vitro transducin activation assays (Reeves et al. 621
1996). Not only did yeast-expressed rhodopsin maintain its ability to respond to light, but the 622
magnitude of signal pathway activation was comparable to many previous studies of mutations 623
expressed in mammalian systems. We were also able to observe rhodopsin mutations that 624
resulted in the activation of dark state rhodopsin, which has previously required sensitive 625
spectroscopic assays using immunoaffinity purified rhodopsin (Liu et al. 2013), or gene knock-in 626
animal models (Zhang et al. 2013). This included providing new data on L125R signaling in the 627
dark, a mutant poorly expressed in mammalian cells which has made previous characterization of 628
function challenging (Stojanovic et al. 2003). Mutations at site L125 have been shown to reduce 629
the thermal stability of rhodopsin (Andres et al. 2001), which could lead to dark noise through 630
thermal isomerization of the bound chromophore (Luo et al. 2011). This mechanism was 631
supported by our finding that L125R dark noise was retinal-dependent in yeast, which could be a 632
result of thermal isomerization. Thus, yeast may serve as a platform for studying difficult to 633
express rhodopsin proteins, allowing for the rapid quantification of downstream G protein 634
activation under various conditions. 635
Mutations known to disrupt rhodopsin folding or stability tended to have incomplete 636
localization on the yeast plasma membrane, similar to mammalian cell expressed rhodopsin. 637
Although there was significant heterogeneity between the investigated mutants, this observation 638
suggests that the biochemical properties of rhodopsin were conserved in yeast, despite known 639
differences in glycosylation patterns (Mollaaghababa et al. 1996). We quantified membrane 640
localization using a novel automated image analysis procedure, which may be useful for studies 641
of other GPCRs and associated pathogenic mutations when expressed in yeast. However, not all 642
human GPCRs are productively expressed in yeast (O'Malley et al. 2009), so these methods 643
should first be validated with the wild-type receptor. 644
The majority of disease-linked rhodopsin mutations that have been identified cause the 645
protein to misfold, which can activate the UPR in the ER, and eventually lead to photoreceptor 646
cell death (Athanasiou et al. 2018). Modulating the UPR has been investigated as a potential 647
treatment for RP (Tam et al. 2010, Chiang et al. 2012a, Parfitt et al. 2014), but the effect on UPR 648
upregulation had only been determined for three rhodopsin mutations (Lin et al. 2007, Kunte et 649
al. 2012, Marsili et al. 2015). Determining UPR upregulation by mutant rhodopsin has 650
previously required microscopy, immunoblot, and qPCR methods (Kunte et al. 2012, Marsili et 651
al. 2015). We took advantage of an engineered yeast strain that possesses a reporter linked to the 652
IRE1 UPR pathway, which is the only one of three UPR pathways that is conserved between 653
mammals and yeast (Kimata and Kohno 2011). This yeast-based reporter of UPR activity 654
enables the use of flow cytometry, a more simple and high-throughput method, and offers the 655
advantage of measuring UPR upregulation directly in live cells. We found that upregulation of 656
this pathway was associated with certain rhodopsin mutants known to misfold or with reduced 657
stability, which may be predictive of the molecular mechanism contributing to retinal 658
degeneration. 659
However, yeast do not contain the PERK and ATF6 UPR pathways found in mammalian 660
cells (Kimata and Kohno 2011). Thus, although the yeast-based methods provide insight into 661
UPR upregulation, these studies would need to be combined with mammalian cells to better 662
understand how all UPR pathways may be affected by rhodopsin and other GPCR mutations. 663
This may also explain why nine of the fifteen mutants that are believed to misfold did not cause 664
an increase UPR activation in yeast, suggesting significant heterogeneity between rhodopsin 665
mutations. Determining a missense mutation’s contribution to UPR upregulation is highly 666
relevant to pharmacogenomics, as an inability for rhodopsin to respond to light may not 667
necessarily indicate the pathogenic mutation can be rescued by UPR modulation. 668
Overall, many known rhodopsin phenotypes were recapitulated in yeast, a requirement 669
for any assay of human gene function seeking to determine the clinical relevance of patient 670
derived missense mutations (Amendola et al. 2016). There are also important differences to 671
consider when comparing our yeast-based assays to mammalian cell-based assays. Importantly, 672
when a lack of signaling is observed in yeast it is difficult to separate rhodopsin mutants that 673
misfold, from mutants that fold properly but do not productively activate the downstream 674
pathway. Although our quantified microscopy data supported the notion that rhodopsin mutants 675
with inconsistent plasma membrane expression in yeast are also mutants known to be unstable or 676
misfolded, unique aspects of yeast cellular machinery or post-translational processing may 677
influence these mutants. Results from the R135G mutation highlight these differences, where this 678
mutant activated the UPR in yeast, but does not aggregate in the ER of mammalian cells 679
(Chuang et al. 2004). 680
The signaling phenotypes of the G90D and A292E mutants in yeast also differed from the 681
reported constitutive activity in vitro when using purified protein (Gross et al. 2003). That 682
A292E signaled higher and G90D lower than expected, suggests that constitutive activity in 683
yeast is dependent on cellular conditions which would not be revealed in an in vitro assay using 684
purified protein, such as a renewing supply of both rhodopsin and G protein. It is interesting to 685
note, however, that A292E has the highest constitutive activity reported of a CSNB-associated 686
mutation in vitro (Gross et al. 2003), which fits the trend observed in yeast. That light-dependent 687
signaling in yeast was affected by the V104I, F220L, and V345M mutations was unexpected, 688
which provides interesting new data for these previously uncharacterized mutants that should be 689
followed up in mammalian cell-based assays. Thus, these yeast-based methods provide 690
complimentary but independent data to traditional mammalian-cell and in vitro biochemical 691
techniques, offering a unique perspective on rhodopsin structure and function in a cellular 692
context. 693
694
Characterizing Novel Rhodopsin Mutations with Yeast 695
Determining mutant function rapidly and accurately has become increasingly important 696
with the rise of whole genome sequencing, and the ever expanding rise in gene mutations with an 697
unknown impact on human health. Rhodopsin mutations linked to inherited retinal disease have 698
been used as examples of how many gene mutations discovered in patients are rarely 699
characterized, and that the molecular basis for pathology is poorly understood (Chiang and Gorin 700
2016, Davies 2014). Animal models and traditional in vitro assays provide detailed information, 701
but they have not kept pace with the hundreds (>350) of rhodopsin mutations identified to date. 702
This is true of many other genetic diseases, but new methods of functional characterization are 703
helping to address this (Starita et al. 2017), including using yeast-based assays (Sun et al. 2016, 704
Yang et al. 2017). 705
We investigated the use of yeast to characterize novel and understudied pathogenic 706
mutations, and compared to clinical data for patients with varying severity of RP. Our yeast-707
based approaches predicted severe phenotypes for the V81Δ and A164 mutations, which 708
included determinations of their light-activation, subcellular localization, and UPR upregulation. 709
By combining yeast and mammalian cell-based assays, the relative severity of these mutations 710
was revealed, as compared with clinical phenotypes measuring decline in visual function in 711
patients. The V81Δ rhodopsin mutation showed the most severe phenotype in our combined 712
assays and the most extensive visual deterioration clinically, in contrast to A164V, which had the 713
mildest phenotype both clinically and experimentally, and A164E, which was found to be 714
intermediate. 715
The difference in severity for the two missense mutations found at site 164 highlights the 716
heterogenic nature of retinitis pigmentosa, and the importance of characterizing individual 717
disease phenotypes. These results indicate that yeast-based approaches could be useful not only 718
for investigating the molecular basis of retinal disease, but also for better prediction of mutation 719
pathogenicity, to help improve the accuracy of prognoses for patients associated with specific 720
mutations in rhodopsin. Integrating the results of both UPR and light-activation assays (Figure 721
S10) may also help determine the molecular mechanism of disease, to differentiate between 722
mutations that cause severe misfolding, versus disruptions in retinal binding or stability that 723
prevent activation but do not cause cell stress. 724
Recent successes in gene therapy for inherited retinal disease means that mutation 725
classification is of utmost importance, to determine if such therapy is required (U.S. Food and 726
Drug Administration 2017). This issue is particularly important to address for degenerative 727
diseases, such as inherited retinal disease, where early intervention is crucial. A method to 728
determine the functional consequences of mutations throughout rhodopsin, rapidly and 729
accurately, would therefore be highly beneficial. 730
The methods presented here could also extend to functionally characterizing mutations of 731
many other GPCRs. Indeed, although over 30 human GPCRs have been functionally linked to 732
the yeast mating pathway, no previous yeast-based study has focused on direct functional 733
characterization of human GPCR mutations. As discussed in a recent review of GPCR 734
pharmacogenomics, characterizing GPCR mutations could lead to a better understanding of 735
disease and drug responses in patients (Hauser et al. 2018). Missense mutations that modulate 736
interactions with downstream signaling and regulatory proteins are known to play a role in this, 737
so assays which accurately reflect GPCR function and interactions in a cellular context, such as 738
the yeast assays presented here, will be key to understanding the impact of GPCR mutations on 739
human health. 740
741
Figures 742
743
Figure 1 Representation of the Engineered Mating Pathway. Rhodopsin activation was 744
functionally coupled to the expression of a fluorescent reporter protein, mCherry, utilizing the 745
mating-responsive promoter pFUS1. Modifications to the mating pathway included the knockout 746
of negative regulator Sst2, the gene encoding Far1 which halts cell growth in the wild-type 747
mating pathway, and the endogenous mating pathway GPCR Ste2. The chimeric G alpha protein 748
(Gpa1-Gαt) contains the 5 C-terminal amino acids of the G alpha subunit of human transducin 749
(Gαt). 750
751
Figure 2 Light-Dependent Activation of the Mating Pathway. Human rhodopsin was found to 752
activate the mating pathway only in response to light, requiring the presence of retinal 753
chromophore. Adding retinal after each hourly light exposure improved the overall response 754
approximately 1.6-fold. Incubating with the same concentration of retinal but keeping the culture 755
in the dark did not result in mating pathway activation. “Vector” denotes yeast transformed with 756
a plasmid not containing the rhodopsin gene. Data points represent results of four individual 757
colonies, each in a 5 mL culture. Error bars represent standard deviation. 758
759
Figure 3 Characterization of Rhodopsin Light-Dependent Function. (A) Response to light 760
from yeast-expressed rhodopsin mutants, indicating a similar magnitude of response as the 761
mammalian cell-expressed protein. “WT” denotes wild-type human rhodopsin. Yeast data points 762
represent results of nine individual colonies, each in a 600 μL culture, minus the mCherry 763
fluorescence of the same strain transformed with empty plasmid control (Vector), and 764
normalized to wild-type. * P < 0.05 vs WT or between indicated mutants. (B) Difference spectra 765
of mammalian cell-expressed rhodopsin mutants in response to light. The peak at 500 nm 766
indicates a light-dependent response. 767
768
Figure 4 The Light-Activated Signal Transduction of Pathogenic Rhodopsin Mutants in 769
Yeast. (A) Missense mutations that intrinsically disrupt rhodopsin stability, or that indirectly 770
affect stability by disrupting a post-translational modification (PTM) motif. Class 3 denotes a 771
unique category of mutations at site R135, which disrupt G protein coupling and lead to 772
constitutive endocytosis in mammalian cells. (B) Pathogenic and asymptomatic variants that 773
increase or do not disrupt rhodopsin activation by light. “WT” denotes wild-type human 774
rhodopsin. “Vector” denotes yeast transformed with a plasmid not containing the rhodopsin 775
gene. “arRP” denotes autosomal recessive retinitis pigmentosa. Data points represent results of 776
nine individual colonies, each in a 600 μL culture, minus the mCherry fluorescence of Vector, 777
and normalized to wild-type. Error bars represent the 95% CI, * P < 0.05 vs WT. 778
779
780
781
782
783
Figure 5 Representative Subcellular Localization of Rhodopsin Mutants (A) Comparative 784
subcellular localization of rhodopsin mutants in SK-N-SH and yeast cells. “PM merge” indicates 785
fluorescence of a plasma membrane marker, merged with GFP-tagged rhodopsin. Images 786
represent maximal projections, with scale bars representing 30 μm and 5 μm respectively. (B) 787
Quantified localization of GFP-tagged rhodopsin mutants on the yeast plasma membrane. Yeast 788
cells with patchy membrane expression of rhodopsin had at least 10% of their cell membrane 789
containing separated patches of rhodopsin. Error bars represent the bootstrapped standard error. 790
791
Figure 6 UPR Activation in Yeast Relative to Wild-Type Rhodopsin. GFP expression is a 792
reporter of UPR upregulation, while RFP is constitutively expressed to help correct for changes 793
in global protein expression. “WT” denotes wild-type human rhodopsin. “Vector” denotes yeast 794
transformed with a plasmid not containing the rhodopsin gene. “Strain” denotes the yJW1200 795
strain not transformed with plasmid and grown in rich media. “arRP” denotes autosomal 796
recessive retinitis pigmentosa. Data points represent results of nine individual colonies, each in a 797
600 μL culture, normalized to wild-type. Boxes extend from the 25th to 75th percentile, the line 798
across the box represents the median value. Bars represent the min and max recorded values. * P 799
< 0.05 vs WT. 800
801
802
Figure 7 Characterization of Novel Rhodopsin Mutants using Yeast. (A) Crystal structure of 803
rhodopsin, highlighting residues V81 and A164 in red. The other rhodopsin mutants 804
characterized in this study are highlighted in cyan, and the approximate location of the 805
membrane is indicated by the dotted line (1U19.pdb). Quantified phenotype and functional 806
assays of V81Δ, A164E, and A164V in yeast (B) Representative subcellular localization, scale 807
bars are 5 μm, (C) quantified consistency of rhodopsin localization on the yeast plasma 808
membrane, (D) light-activated signal transduction, and (E) UPR activation. “WT” denotes wild-809
type human rhodopsin. The number of biological replicates and error bars are identical to 810
previous figures. * P < 0.05 vs WT in all panels unless otherwise indicated. 811
812
Figure 8 Characterization of Novel Rhodopsin Mutants using Mammalian Cell Expression. 813
(A) Comparative subcellular localization of rhodopsin mutants in SK-N-SH cells. “PM” 814
indicates fluorescence of a plasma membrane marker, “ER” indicates fluorescence of a ER-815
specific marker. Scale bars are 30 μm. (B) Difference spectra of mammalian cell-expressed 816
rhodopsin mutants in response to light. The peak at 500 nm indicates a light-dependent response. 817
818
819
820
Figure 9 Clinical Assessment of Patients with Rhodopsin Mutations V81Δ, A164E, and 821
A164V. (A) Goldmann visual fields of the right eye at two time points. Normal fields would 822
reach the gray dotted line. The solid blue line outlines the actual field. The hatched areas are 823
scotoma, i.e. areas of loss in sensitivity. Darker areas refer to denser scotoma. (B) Structural 824
retinal phenotype of the right eye from cases carrying the A164E and V81Δ mutations. Optical 825
coherence tomography (OCT) above showing the different retinal layers. Brackets show area of 826
preserved outer retina; A164E > V81Δ. Unlike for A164E, the OCT of V81Δ shows disturbed 827
lamination of the retina with degenerative cysts, reflecting more advanced disease. The retinal 828
photograph below centered on the posterior pole. Photograph on the right is taken with a wider 829
field camera. ON: optic nerve. The dotted white line indicated the foveal area at the center of the 830
macula. Double white arrow indicates vessel attenuation, while single arrow shows typical 831
pigmentary deposits (few in these cases). The width of the central visual field corresponds to the 832
area of preserved outer retina on the OCT. 833
834
835
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