, Carmen Aguado , Martin Horst and Erwin Knecht D · Natalia Salvador‡\\, Carmen Aguado‡ |,...

$Page 1: , Carmen Aguado , Martin Horst and Erwin Knecht D · Natalia Salvador‡\\, Carmen Aguado‡ |, Martin Horst¶ and Erwin Knecht‡D From the ‡Instituto de Investigaciones Citológicas,$
1

Import of a Cytosolic Protein into Lysosomes by Chaperone-Mediated

Autophagy Depends on its Folding State\\ §

Natalia Salvador‡\\ \\ , Carmen Aguado‡ || ||, Martin Horst¶ and Erwin Knecht‡DD

From the ‡Instituto de Investigaciones Citológicas, Fundación Valenciana de

Investigaciones Biomédicas, Amadeo de Saboya, 4, Valencia 46010, Spain and

the ¶ Faculty Phil. II, University of Basel, Missionstr. 64, CH-4055 Basel,

Switzerland

\This project was supported by the Ministerio de Educación y Ciencia (grants

PB97-1445 and PM98-0041) and Fundació La Caixa (grant 97/131-00).

§N. Salvador and C. Aguado contributed equally to this work.

\\A predoctoral fellow of the Consellería de Cultura Educación y Ciencia de la

Generalitat Valenciana.

||A postdoctoral fellow of the Fundación Bancaixa.

DSend correspondence to:

Erwin Knecht

Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones

Biomédicas, Amadeo de Saboya, 4, 46010-Valencia, Spain.

Tel.: 346-3391250; Fax: 346-3601453; E-mail: [email protected].

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 20, 2000 as Manuscript M001394200 by guest on February 16, 2020

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Running title: FOLDING STATE OF A CYTOSOLIC PROTEIN IMPORTED INTO

LYSOSOMES.

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SUMMARY.

We have analysed the folding state of cytosolic proteins imported in vitro

into lysosomes, using an approach originally developed by Eilers and Schatz,

(Nature 322 (1986), 228-232) to investigate protein import into mitochondria. The

susceptibility towards proteases of mouse dihydrofolate reductase (DHFR),

synthesised in a coupled transcription-translation system with rabbit reticulocytes,

decreased in the presence of its substrate analogue, methotrexate. This analogue

complexes with high affinity with the in vitro synthesised DHFR and locks it into a

protease-resistant folded conformation. DHFR was taken up by freshly isolated rat

liver lysosomes and methotrexate reduced this uptake by about 80%. A chimeric

DHFR protein, which carries the N-terminal presequence of subunit 9 of ATP

synthase preprotein from Neurospora crassa fused to its N-terminus, was taken

up by lysosomes more efficiently. Again, methotrexate abolished the lysosomal

uptake of the fusion protein, which was partially restored by washing of

methotrexate from DHFR or by adding together methotrexate and dihydrofolate,

the natural substrate of DHFR. Immunoblot analysis with-anti-DHFR of liver

lysosomes and of other fractions, isolated from rats starved for 88 h and treated

with lysosomal inhibitors, suggests that DHFR is degraded by chaperone-

mediated autophagy. Competition with ribonuclease A and stimulation by

ATP/Mg2+ and the heat shock cognate protein of 73 kDa show that the lysosomal

uptake of the fusion protein also occurs by this pathway. It is concluded that the

lysosomal uptake of cytosolic proteins by chaperone-mediated autophagy mainly

occurs by passage of the unfolded proteins through the lysosomal membrane.

Therefore, this mechanism is different from protein transport into peroxisomes, but

similar to the import of proteins into the endoplasmic reticulum and mitochondria.


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INTRODUCTION.

Proteins are continuously being degraded by both lysosomal (1) and non-

lysosomal (2) proteases. Lysosomes, which are found in almost all eukaryotic

cells, participate in intracellular protein degradation by various mechanisms (1):

endocytosis, crinophagy, direct conversion of endoplasmic reticulum (ER)1

cisternae into lysosomes, macroautophagy, microautophagy and a selective

pathway for the uptake and degradation of cytosolic proteins mediated by the heat

shock cognate protein of 73 kDa (hsc73), also known as chaperone-mediated

autophagy.

Endocytosis is the degradative route followed by extracellular proteins,

which are either recognised by specific receptors or simply trapped by nonspecific

uptake (3). This is also the degradative pathway followed by plasma membrane

proteins which, unlike the LDL or the transferrin receptors, do not recycle back to

the plasma membrane for reuse. By crinophagy (4), proteins originally destined for

secretion are delivered to lysosomes, when the demands for these proteins

decline, by a process involving fusion of secretory granules with endosomes

and/or lysosomes instead of with the plasma membrane. Also, proteins in transit

through the ER, as well as membrane and luminal resident proteins of the ER,

can be degraded, under certain conditions, by a direct conversion of cisternae of

the transitional part of the ER into lysosomes (5). In macroautophagy, or classical

autophagy, large areas of cytoplasm, typically including whole organelles, are

sequestered by a segregating structure and degraded by lysosomes (6, 7).

Microautophagy is a degradative route whereby portions of cytoplasm, including

certain organelles such as peroxisomes and/or cellular components down to the

level of macromolecules, are directly internalised into the lysosomal matrix by

various modifications of the lysosomal membrane which produce intralysosomal

vesicles (8). Finally, in serum-deprived confluent fibroblasts, a selective pathway

was described for the degradation of ribonuclease A (RNase A) which required

the pentapeptide sequence KFERQ, hsc73 and ATP/Mg2+ (9).

Since its original discovery, the chaperone-mediated autophagic pathway

for the uptake and degradation of cytosolic proteins has been found to be also


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operative in rat liver, especially under long-term starvation (10, 11), and in kidney

and heart, but not in brain, testes and skeletal muscle (10). This selective uptake

of cytosolic proteins requires KFERQ-like sequences (9), ATP/Mg2+ and a

cytosolic (12) and an intralysosomal (13, 14) hsc73. In addition, the lysosomal

membrane glycoprotein lamp-2a was suggested as a receptor for this pathway

(15). The pathway has been also, at least partially, reconstituted in vitro with

lysosomes from rat liver (16, 17) and from human fibroblasts (12, 18), as well as

with yeast vacuoles (19). In this latter case, the relationship of the observed

uptake with the in yeast well established uptake of cytosolic proteins into vesicular

intermediates, which is followed by fusion with the vacuolar membrane (20, 21),

remains to be clearly demonstrated. From all this work, it appears that the

selective transport of proteins into mammalian lysosomes occurs by a process

which resembles, in some respects, the import of proteins synthesised on

cytosolic ribosomes into other cell compartments, such as mitochondria,

chloroplasts and the various types of microbodies (peroxisomes, glycosomes,

hydrogenosomes and glyoxysomes)(22-25).

Uptake of cytosolic proteins into lysosomes for degradation by the

chaperone-mediated autophagic pathway requires the movement of the protein

across the lysosomal membrane. Proteins can be transported into organelles

either in an unfolded (as for example occurs in mitochondria, chloroplasts or

endoplasmic reticulum, see (22-24) for review) or in a folded conformation (as is

the case, for example, for proteins transported into peroxisomes or into the

thylakoid membranes of the chloroplast, see (25, 26) for review). However, the

conformation of the protein as it passes from the cytosol into the lysosomal lumen

is still unknown. Since the original work of Eilers and Schatz (27), the cytosolic

enzyme dihydrofolate reductase (DHFR) has been used to investigate the

conformation of proteins as they pass across membranes, taking advantage of the

fact that its conformation can be stabilised by complexing it with methotrexate.

Here, we have analysed the effect of methotrexate on the uptake by isolated rat

liver lysosomes of the cytosolic enzyme DHFR synthesised in vitro in a coupled

transcription-translation system with rabbit reticulocytes. From the obtained


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results we conclude that DHFR passes through the lysosomal membrane mostly

in an unfolded conformation.


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EXPERIMENTAL PROCEDURES.

Materials.

Metrizamide (grade I), trypsin, elastase, trypsin inhibitor, elastatinal,

phenylmethylsulfonyl fluoride, chymostatin, chloroquine, methotrexate,

dihydrofolate, Triton X-100, 3-[N-morpholino] propanesulfonic acid (MOPS), OsO4,

ATP, creatine phosphate, creatine phosphokinase, ATP-agarose, p-nitrophenol-N-

D-acetyl glucosaminide, 4-methylumbelliferyl-2-acetamido-2-deoxy-β-D-

glucopyranoside, goat anti-mouse IgM- and goat anti-mouse IgG (H+L)-alkaline

phosphatase conjugates, 5-bromo-4-chloro-3-indolyl phosphate, nitro blue

tetrazolium, RNase A and ribonuclease S-protein (RNase S-protein) were from

Sigma-Aldrich Química S.A. (Madrid, Spain). Leupeptin was from Peptide Institute

(Osaka, Japan). Antibodies against hsc73 were obtained from clone 13D3 (mouse

immunoglobulin M) from Maine Biotechnology Services (Portland, ME). Anti-

DHFR-polyclonal antibodies were raised against 6-His-tagged mouse DHFR

cloned in pQE16 (Qiagen, Hilden, Germany) and overexpressed in Escherichia

coli. Sodium-deoxycholate, uranyl acetate, proteinase K and sucrose were from

Merck (Darmstadt, Germany). Bovine serum albumin (fraction V) was from

Boehringer (Mannheim, Germany). Acrylamide/Bis 29:1 was from Bio-Rad

(Richmond, CA). Sodium dodecyl sulfate was from Serva (Heidelberg, Germany).

Cellulose nitrate paper (0.45 µm) was from Schleicher and Schuell (Dassel,

Germany). Glutaraldehyde was from Tousimis (Rockville, MD). Epon (Poly/Bed

812 resin) was from Polysciences (Warrington, PA). TNT-coupled rabbit

reticulocyte lysate system was from Promega (Madison, WI). Tran35S-label (70%

L-methionine) was from ICN Pharmaceuticals, Inc. (Irvine, CA) and 3H-leucine (44

Ci/mmol) was from NEN (Boston, MA). Other reagents were of the best analytical

quality available.

In vitro transcription and translation of mouse DHFR, a fusion protein between the

presequence of F0-ATPase subunit 9 and mouse DHFR and human

glyceraldehyde-3-phosphate dehydrogenase.


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A recombinant pSP65 plasmid containing a BamHI-HindIII fragment of 600

base pairs, carrying the full-length mouse DHFR with 6 histidines on its C-

terminus, was used for the expression of DHFR. Also, recombinant pGEM3

plasmid (28) containing a SmaI fragment of 777 base pairs, which includes the

fusion protein between the presequence and three amino acid residues of the

mature part of subunit 9 of the F0-ATP synthase of Neurospora crassa and the

mouse full length coding region of DHFR was used. This fusion protein is called

hereafter Su9-DHFR for brevity. For in vitro translation of glyceraldehyde-3-

phosphate dehydrogenase (GAPDH), a recombinant pSP64 plasmid, designed

pIC328, containing a PstI-BamHI fragment of about 1400 base pairs, which

includes the full-length human GAPDH coding region plus additional 5´- and 3´-

non-translated sequences (16), was used.

In vitro transcription and translation of the various DNAs with the TNT-

coupled rabbit reticulocyte system was carried out following the manufacturer´s

instructions.

Proteolytic susceptibility measurements.

Incubations were carried out at 37 ºC in a final volume of 20 µl. Assays

contained 5 µl of the standard synthesis reaction, with and without 250 nM

methotrexate, 0.1 M triethanolamine buffer, pH 7.6, and elastase (1% in terms of

protein and referred to the protein in the rabbit reticulocyte lysate, 111.5 mg

protein/ml). At the times indicated, portions were taken, 50 µM elastatinal was

added, and the samples were subjected to sodium dodecyl sulfate polyacrylamide

gel electrophoresis (SDS-PAGE) and fluorography. Experiments were also carried

out with trypsin and trypsin inhibitor with similar results (data not shown).

Preparation of lysosomes.

24 h-fasted male Wistar rats (Interfauna Ibérica S.A., San Feliu de Codines,

Spain) were used throughout. All rats were fed ad libitum for at least 7 days before

the experiments began. In some experiments, rats were treated with leupeptin (2


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mg/100 g weight, intraperitoneally, 1.5 h before sacrifice). Lysosomes were

obtained from a light mitochondrial fraction in a discontinuous metrizamide

gradient by a procedure based on that originally developed by Wattiaux et al. (29).

Rats (200-250 g) were anaesthetised with ether and decapitated. 10 g of rat liver

were used and homogenate (7 ml of chilled 0.25 M sucrose/g liver) was filtered

through cheese-cloth and centrifuged in a Sepatech Biofuge 28RS (Heraeus

Sepatech GmbH, Osterode, Germany) at 4,800 x g for 10 min and the supernatant

at 17,000 x g for 10 min. After resuspension and washing once, the sediment (a

light mitochondrial-lysosomal fraction), suspended in 57% metrizamide, was

loaded on the bottom of a discontinuous metrizamide gradient (adjusted to pH

7.0). The discontinuous metrizamide gradient consisted of the following layers:

19.8% (top layer, 3.9 ml), 26.3% (3.5 ml), 32.8% (2.2 ml) and 57.0% (2.3 ml).

Centrifugation in the metrizamide gradient was for 90 min in an SW 40 Ti

rotor (Beckman) at 141,000 x g. Lysosomes were collected from the most upper

layer and the 26.3-19.8% interface, diluted at least five times with 0.3 M sucrose

and sedimented at 37,000 x g for 10 min in a Sorvall centrifuge (rotor SS-34).

Mitochondria were obtained from the 57-32.8% interface (16). All procedures were

carried out at 0-4 ºC. After a quick wash, lysosomes were carefully resuspended

in 10 mM MOPS, pH 7.2 and 0.3 M sucrose to a concentration of about 6 mg

lysosomal protein/ml and immediately used for the uptake experiments. Yields

were 0.3-0.5 mg of lysosomal protein per g of liver. The appearance of the

lysosomes by electron microscopy was not changed during the various

incubations used here. The integrity of the lysosomal membranes was also

estimated by measuring the latency of the lysosomal enzymes β-hexosaminidase

and β-N-acetyl glucosaminidase (11). It was >94% at the end of the incubations.

In some experiments, two lysosomal fractions were prepared by

centrifuging separately the top layer and the 26.3-19.8% interface of the

metrizamide gradient as described (14). These fractions (referred to hereafter as

HSC+ and HSC- lysosomes) contained or did not contain, respectively, hsc73

within the lysosomal lumen (14 and data not shown).


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Cell-free assay of transport of cytosolic proteins into lysosomes.

Freshly isolated lysosomes (60 µg protein), treated or not with chymostatin

(see below), were incubated, for 10 min at 30 ºC in a final volume of 40 µl, in 0.3

M sucrose/10 mM MOPS buffer (pH 7.2), containing an ATP-regenerating system

(final concentrations: 10 mM MgCl2, 10 mM ATP, 2 mM phosphocreatine and 50

µg/ml creatine phosphokinase) (medium S) with the various in vitro synthesised

proteins (10 µl of the standard synthesis reaction, except where indicated), treated

or not with 250 nM methotrexate. Although lysosomes (11, 13, 14) and rabbit

reticulocyte lysates (data not shown) do contain hsc73 in enough amounts for

lysosomal transport, in most experiments additional hsc73 (5 µg/ml, final

concentration) was added to the incubation mixture. Chymostatin, when used,

was added to the lysosomes at 0 ºC for 10 min at three times its final

concentration (30 µM) and then diluted threefold with incubation buffer containing

the in vitro synthesised proteins. Experiments were also carried out with 10 mM

(final concentration) chloroquine with similar results (data not shown). Samples

were centrifuged in a Sepatech Biofuge 28 RS (rotor HFA 22.1) at 26,000 x g for 5

min at 4 ºC, the pellets were quickly washed once and pellets and supernatants

were subjected to SDS-PAGE and fluorography. In some experiments, a treatment

of proteinase K (20 µg per tube) in 0.3 M sucrose/MOPS buffer (pH 7.2) of the

washed sediments was carried out, with or without 1% Triton X-100, for 10 min at

0 ºC. After addition of 2 mM phenylmethylsulfonyl fluoride, samples were

centrifuged as above and the pellets and the supernatants were subjected to

SDS-PAGE and fluorography.

In experiments with HSC+ and HSC- lysosomes, prior to the standard

incubation with the in vitro synthesised proteins and the following treatments,

lysosomes were preincubated for 5 min at 30 ºC with 0.3M sucrose/10 mM MOPS

buffer (pH 7.2) containing or not hsc73 and/or the ATP-regenerating system.

Competition experiments between RNase A and RNase S-protein and the in vitro

synthesised proteins, were carried out as described (17).


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Proteolysis measurements with freshly isolated lysosomes.

Chinese hamster ovary (CHO) cells were grown as described (30). They

were metabolically labelled for 48 h with 25 µCi/dish 3H-leucine and a cytosolic

fraction was prepared as described previously (31). Freshly isolated lysosomes

(60 µg of protein) were incubated for different times in medium S with the 3H-

labelled cytosolic proteins from CHO cells (10 µg protein with a specific activity of

about 10,000 dpm/µg protein) and 10 µl of the reticulocyte lysate transcription-

translation mixture, with or without DHFR plus 250 nM methotrexate, in a total

volume of 100 µl. At 0, 5, 10, 20, 30 and 40 min incubation, aliquots were taken

and mixed with 500 µl of 10% (w/v) trichloroacetic acid to terminate the reaction. 3

mg bovine serum albumin were added to each aliquot as carrier protein and the

samples were incubated for 10 min on ice and then pelleted by centrifugation.

Under these conditions, non-degraded proteins are precipitated, whereas

proteolytic fragments remain soluble. To control for the integrity of the isolated

lysosomes during the incubation period (16), lysosomes were incubated in parallel

as above but without the 3H-labelled soluble proteins. At 5, 10, 20, 30 and 40 min,

lysosomes were removed by centrifugation and supernatants were incubated with3H-labelled cytosolic proteins for the same times (5, 10, 20, 30 and 40 min,

respectively). The reaction was stopped with trichloroacetic acid plus bovine

serum albumin as above. The radioactivity of the acid-soluble and acid-insoluble

material (dissolved in 0.2 M NaOH containing 0.4% sodium deoxycholate) was

determined by liquid scintillation counting. Degradation was expressed as

percentage of the initial acid-insoluble radioactivity remaining at the different

incubation times.

General

DNA sequencing was performed using a 377 Automated DNA sequencer of

Applied Biosystems (Foster City, CA). Protein concentration was measured by a

modification, with sodium-deoxycholate (32), of the Lowry et al. (33) method and


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using bovine serum albumin as the standard. Hsc73 was purified from rat liver

cytosol by ATP-agarose affinity chromatography (34). Cytosol was the supernatant

of three successive centrifugations of rat liver homogenates at 2,500 x g, 10 min,

17,000 x g, 10 min and 155,000 x g, 60 min in a Beckman L5-65 centrifuge, Ti-

70.1 rotor. SDS-PAGE was done according to Laemmli (35) and gels were used

for fluorography. The radioactivity associated with the various proteins in the

autoradiograms was quantified by phosphorimager analysis using a FLA-2000

image analyser and Science Lab 98 Image Gauge ver. 3.11 software from

FujiFilm España S.A. (Barcelona, Spain). Statistical analyses were carried out

with Student´s t test. Immunoblotting procedures were carried out as described

previously (14, 16). Lysosomal fractions, treated or not with methotrexate, were

fixed and embedded in Epon for conventional electron microscopy by standard

procedures (36). All data shown are representative results of at least three

separate experiments.


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RESULTS.

To test the effect of methotrexate on the conformation of DHFR, aliquots of

in vitro synthesised [35S]-labelled-DHFR, treated or not with methotrexate (250

nM), were incubated with elastase for increasing time periods (Fig. 1A). In the

absence of methotrexate (lanes 2-5), and under the conditions of the experiments,

DHFR was degraded, but in its presence (lanes 6-9), DHFR was protected from

elastase by the bound methotrexate.

To follow the import of DHFR into lysosomes, we mixed [35S]-labelled

DHFR with freshly isolated rat liver lysosomes in an isotonic medium at pH 7.2

(see Experimental Procedures) in the absence or in the presence of chymostatin

(to inhibit intralysosomal proteolysis) (Fig. 1B). This in vitro assay, to monitor the

uptake of cytosolic proteins into rat liver lysosomes, has been described in detail

(e.g. 16, 17). As it is the case with other proteins previously investigated, we also

found that, after incubation of the protein with the lysosomes, DHFR was

associated with the washed lysosomal pellets (lanes 2 and 5), especially in the

presence of chymostatin (lane 2). To eliminate the protein which is simply bound

to the external surface of the lysosomal membrane, we used a treatment with an

externally added protease (e.g. proteinase K, see Experimental Procedures). In

the presence of chymostatin, but not in its absence, part of the protein which was

untreated with methotrexate was protected from proteinase K (Fig. 1B, compare

lanes 3 and 6). In the presence of Triton X-100, DHFR associated with lysosomes

was degraded by proteinase K (lane 4). These results indicate that, in the absence

of methotrexate, DHFR is taken up into lysosomes where it is degraded by

lysosomal cathepsins unless chymostatin is present. When the same

experiments were carried out in the presence of methotrexate (Fig. 1C), almost no

uptake of DHFR could be detected, independently of the presence of the inhibitor

of lysosomal proteases. Thus, under the conditions of the experiment, there were

almost no difference in the amount of DHFR associated to the washed lysosomal

pellets with or without chymostatin (lanes 2 and 5), and proteinase K released

from the lysosomal membrane the associated DHFR (lanes 3 and 6).


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Methotrexate neither affected the activity of elastase (as observed by

digestion of albumin by elastase in the presence of 250 nM methotrexate, data not

shown), nor the lysosomes which were used in these experiments. Thus, the

electron microscopic appearance of lysosomes incubated in the presence of

methotrexate for 10 min was not different from that of lysosomes incubated in

parallel but without methotrexate (Fig. 2). Also, the latency of the lysosomal

enzymes β-hexosaminidase and β-N-acetyl glucosaminidase was not modified by

the methotrexate treatment (data not shown). Finally, Fig. 3 shows that the

association of DHFR and methotrexate neither affects the lysosomal integrity

(compare s—s with ∆---∆), nor the proteolytic activity of lysosomes towards an

extract of cytosolic proteins (compare n—n with o---o).

Moreover, when a DHFR-unrelated protein (GAPDH) was used in similar

assays to those shown in Fig. 1 it was found that methotrexate neither modified

the proteolytic susceptibility of GAPDH towards elastase (data not shown) nor the

transport of GAPDH into lysosomes (Fig. 4).

The use of an exogenously added protease (proteinase K) in the assay

employed in Fig. 1B and C has some inconveniences when used to quantitatively

compare the lysosomal uptake of proteins with very different susceptibilities to

proteases, such as DHFR and DHFR-methotrexate. Thus, proteinase K, at the

conditions needed to eliminate DHFR-methotrexate from the lysosomal

membranes, also affects, to some extent, the lysosomal membrane. This,

together with the higher proteolytic susceptibility of DHFR without methotrexate to

proteinase K, determines that the amount of DHFR transported into lysosomes, in

the absence of methotrexate, might be underestimated. Therefore, to quantify

more accurately the effect of methotrexate on the binding and uptake of DHFR, we

incubated DHFR expressed in the coupled transcription-translation assay with

freshly isolated lysosomes with or without an inhibitor of lysosomal proteases. We

have already shown in previous papers (e.g. 11, 14, 16, 17, 37) that the protein

associated to lysosomes incubated without inhibitors of lysosomal proteases

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these conditions, the internalised protein is degraded (as confirmed also here by

the proteinase K treatment, see Fig. 1B). In the presence of an inhibitor of

lysosomal proteases, part of the protein is insensitive to proteinase K if it is

transported into the lysosomes (Fig. 1B, lane 3). This part represents the protein

which has entered into lysosomes and is not degraded by the lysosomal

proteases (because they are inhibited) and which is also protected from the

exogenously added protease by the lysosomal membrane, unless a detergent

(Triton X-100) is added. Therefore, for a specific protein, the protein associated to

lysosomes in the presence of inhibitors of lysosomal cathepsins (here

chymostatin) represents both the protein bound to the lysosomal membrane

(released by proteinase K) and the internalised protein (insensitive to proteinase

K). Thus, it is possible to quantify the protein which has been taken up by

lysosomes by subtracting the protein associated to lysosomes in the presence

(protein bound to the lysosomal membrane and taken up by lysosomes) and in

the absence (protein bound to the lysosomal membrane) of inhibitors of lysosomal

proteases (11, 14, 16, 17, 37). Table 1 (two upper lines) shows that while the

binding of DHFR to the lysosomal membranes, with or without methotrexate, is

similar, the uptake of DHFR by lysosomes occurs much more efficiently in the

absence of methotrexate.

DHFR is not quite efficiently incorporated into lysosomes (see Table 1, two

upper lines), in agreement with previous observations from Dice´s lab (38, 39).

Therefore, we tested a chimeric DHFR protein (Su9-DHFR) with the N-terminal

presequence of the precursor polypeptide from Neurospora crassa ATP synthase

subunit 9 fused to the N-terminus of DHFR (see Experimental Procedures). The

fusion protein was quite efficiently incorporated into lysosomes (see below),

probably because this presequence contains two possible additional KFERQ-like

sequences (see Discussion). When this construct was incubated with elastase for

increasing time periods (Fig. 5A), we found that, in the absence of methotrexate

(lanes 2-5), DHFR was completely degraded. However, in the presence of

methotrexate (lanes 6-9), the DHFR moiety was protected from the elastase

attack by the bound methotrexate. Here we found, as also observed by others


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(27), that the protease cleaves off the unprotected pre-sub 9 extension, because it

is in an unfolded state, leaving the DHFR moiety intact, because it is protected by

the bound methotrexate. The effect of methotrexate on the binding and uptake by

freshly isolated rat liver lysosomes of Su9-DHFR was quantified in four different

experiments similar to that shown in Fig. 5B (Table 1, two lower lines). These

results show again that while the binding of DHFR to the lysosomal membranes,

with or without methotrexate, is similar, the uptake of DHFR by lysosomes occurs

five times more efficiently in the absence of methotrexate. In both cases, the

uptake efficiency of the fusion protein was higher than when DHFR alone was

used. These results confirm the importance of an unfolded conformation for the

transport of DHFR.

To make sure that Su9-DHFR-methotrexate bound to the lysosomal

membrane was on the lysosomal import pathway, we tried to reverse the effect of

methotrexate, washing the drug from Su9-DHFR bound to the lysosomal

membrane (Fig. 6). During this procedure, part of the membrane-bound Su9-DHFR

was apparently released and there was also some degradation of the imported

Su9-DHFR. However, it was clear that partial removal of methotrexate from Su9-

DHFR bound to the lysosomal membrane led to an increased import of the fusion

protein (Fig. 6A, lanes 4 and 5, compare with lanes 6 and 7, where methotrexate

was present, compare also in Fig. 6B, uptake bars in mtx (+/-) and mtx (+/+)). This

import represents about one third of the total import of the fusion protein subjected

to two successive incubations without methotrexate (Fig. 6A, lanes 2 and 3,

compare also in Fig. 6B, uptake bars in mtx (+/-) and mtx (-/-)).

Dihydrofolate, the natural substrate of DHFR, binds to DHFR with lower

affinity than methotrexate. Thus, we reasoned that DHFR-dihydrofolate could

unfold easier than DHFR-methotrexate. As shown in Fig. 7, dihydrofolate had a

much less protective effect than methotrexate on the proteolysis of Su9-DHFR by

elastase (lanes 4 and 5 in Fig. 7A and +dhf -mtx in Fig. 7B, compare with lanes 6

and 7 in Fig. 7A and -dhf +mtx in Fig. 7B). When methotrexate and dihydrofolate

were used together (lanes 8 and 9 in Fig. 7A and +dhf +mtx in Fig. 7B), Su9-


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DHFR was protected to a lower extent than with methotrexate alone, suggesting

competition of dihydrofolate and methotrexate for the binding site in DHFR. The

same results where obtained with DHFR without the N-terminal presequence of

the precursor polypeptide of subunit 9 of ATP synthase (data not shown).

To find out whether there is a correlation between the folding state of DHFR

and its lysosomal import, the effect of competition of dihydrofolate with

methotrexate on lysosomal import of Su9-DHFR was investigated. Without

methotrexate, dihydrofolate alone reduces the uptake of Su9-DHFR by about 50%

(Fig. 8A compare lanes 4-7 and lanes 2 and 3, compare also in Fig. 8B, + dhf and

- dhf uptake bars). This is probably because dihydrofolate keeps DHFR folded,

although less efficiently than methotrexate (see Fig. 7 and Fig. 8C and D). When

dihydrofolate was used together with methotrexate, it was found that the

lysosomal uptake of DHFR increased slightly (about 2 times) when compared

with methotrexate alone (compare, in Fig. 8C, lanes 4-7 and lanes 2 and 3,

compare also in Fig. 8D, + dhf and - dhf uptake bars). These results indicate that

dihydrofolate and methotrexate compete for the binding site of DHFR and that

dihydrofolate binding keeps DHFR into a more relaxed conformation, as

suggested also by the proteolytic susceptibility observations (Fig. 7). All these

observations stress again the importance of an unfolded conformation for an

efficient lysosomal transport of a cytosolic protein.

These experiments demonstrate that the folding state of both DHFR and

Su9-DHFR affects their uptake by isolated lysosomes. Although this experimental

system has been previously shown to reproduce the chaperone-mediated

autophagic pathway (11-15, 17, 18, 31, 37, 40), the following experiments were

carried out to show more clearly that both proteins are targeted to lysosomes by

this pathway. It is known that in rat liver prolonged starvation (64-88 h) activates

the chaperone-mediated autophagic pathway while reducing the activity of other

lysosomal pathways (10, 11). Therefore, we reasoned that if DHFR follows in vivo

the chaperone-mediated autophagic pathway it should be possible to detect, in

rats subjected to prolonged starvation, DHFR within liver lysosomes whose

proteases are inhibited. Thus, we isolated several liver fractions from rats starved


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for prolonged periods (88 h) which were injected or not with the lysosomal

cathepsins inhibitor leupeptin (Fig. 9A). As expected, DHFR was detected, by

Western blot with an antibody towards DHFR, in whole homogenates (not shown)

and in the cytosol of both leupeptin-treated and untreated 88 h-starved rats (Fig.

9B, lanes 1 and 4). DHFR was not detected in mitochondria and in lysosomes

from untreated rats (Fig. 9A, lanes 2 and 3). However, it was detected in

lysosomes (Fig. 9B, lane 6), but again not in mitochondria (Fig. 9A, lane 5) of

leupeptin-treated rats (i.e. it was found in lysosomes with inhibited proteases).

Moreover, in leupeptin-treated rats, the lysosomal associated protein was partially

resistant to a proteinase K treatment which, under the same conditions,

completely degrades DHFR in the cytosol (Fig. 9C, compare lines 5 and 2,

respectively). Therefore, it appears that in liver of rats starved for 88 h, a condition

under which the chaperone-mediated autophagic pathway is activated, at least

part of the DHFR molecules are transported from the cytosol into lysosomes.

Similar experiments, carried out with rats starved for 20 h, showed that DHFR was

also found within isolated lysosomes from leupeptin-treated rats, but in relatively

smaller amounts as compared to lysosomes isolated from the liver of rats starved

for 88 h (data not shown). It should be noticed that in livers from rats starved for 20

h, although the chaperone-mediated autophagic pathway is also operative (11),

the macroautophagic pathway, which non-selectively degrades all kind of

proteins, is strongly activated (1, 6-8, 11).

Su9-DHFR is a chimeric protein which does not exist in vivo and which, in

the presence of mitochondria (i.e. under in vivo conditions), would be mainly

targeted to these organelles. Although this construct was used here merely as a

model to confirm the observations with DHFR, we carried out experiments to

investigate if the lysosomal uptake of this protein also occurs by chaperone-

mediated autophagy.

In a long series of experiments (reviewed in 9, 40) Dice´s laboratory

showed, both in vivo and in vitro, that RNase A (which contains a KFERQ

sequence at amino acids 7-11) follows the chaperone-mediated autophagic


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pathway of protein degradation. The uptake of RNase A and of other cytosolic

proteins by the chaperone-mediated autophagic pathway requires both ATP/Mg2+

and cytosolic and lysosomal hsc73. Although lysosomes (11, 13, 14) and rabbit

reticulocyte lysates (data not shown) do contain hsc73 in sufficient amounts for

lysosomal transport, we separated, on the basis of their density, two different

lysosomal populations (14 and data not shown), one containing hsc73 (HSC+)

and another without this protein (HSC-). As shown in Fig. 10, without additions,

the lysosomal uptake of Su9-DHFR is considerably reduced, but HSC+ lysosomes

are slightly more effective than HSC- lysosomes in taking up the protein.

Preincubation (see Experimental Procedures) of both lysosomal populations with

an ATP-regenerating system and hsc73 increased considerably their uptake of

Su9-DHFR (Fig. 10A, lanes 2 and 5, compare with lanes 1 and 4, compare also in

Fig. 10B, +ATP +hsc73 bars with Control bars). When hsc73 was omitted from

the incubation mixture with the ATP-regenerating system, there was a decrease in

the uptake of Su9-DHFR by HSC- lysosomes (Fig. 10A, lanes 3 and 6, compare

with lanes 2 and 5, compare also in Fig. 10B, +ATP bars with +ATP +hsc73

bars). This effect was also observed with HSC+ lysosomes although it was less

evident. The same results were obtained when DHFR was used (data not shown).

Moreover, and as is the case with RNase A, Su9-DHFR and DHFR are

incorporated by lysosomes isolated from rats starved for 88 h more efficiently than

when using lysosomes from 20-h starved rats (data not shown). All these

observations are similar to those obtained, in similar experiments, with RNase A

(11, 14).

Finally, and in contrast to RNase A, RNase S-protein (amino acids 21-124

of RNase A and, thus, without a KFERQ sequence) is not a substrate of the

chaperone-mediated autophagic pathway (9, 40). Thus, we carried out

competition experiments between RNase A and RNase S-protein with Su9-DHFR

for lysosomal uptake. Increasing amounts of RNase A (Fig. 11A), but not of

RNase S-protein (Fig. 11B), reduced the lysosomal uptake of Su9-DHFR. Similar

results were obtained with DHFR (data not shown). All these results taken

together show that there are common elements in the lysosomal uptake


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mechanisms of DHFR and Su9-DHFR and RNase A, which has been extensively

shown, both in vivo and in vitro (see 9, 40 for reviews), to be degraded by

chaperone-mediated autophagy.


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DISCUSSION.

In eukaryotic cells, cytoplasmic synthesised proteins can enter into their

organelles of residence in either an unfolded conformation (as for example in

mitochondria) or in a folded conformation (as for example in peroxisomes)(22-26,

41-43). The conformation of proteins which enter into lysosomes by chaperone-

mediated autophagy is unknown. We decided to approach this problem using

dihydrofolate reductase (DHFR) and a procedure first developed by Eilers and

Schatz (27) to study the transport of mitochondrial proteins into this organelle.

The mechanism whereby DHFR is degraded in rat liver is unknown.

Immunoblot analysis of rat liver fractions revealed the presence of DHFR in the

cytosol, as expected, and also within lysosomes from leupeptin-treated rats.

Leupeptin enters lysosomes by endocytosis (44) and it has been shown to be very

effective in inhibiting, under in vivo conditions, rat liver lysosomal cathepsins (45,

46). This has allowed the identification of several in vivo substrates of rat liver

lysosomes under a variety of conditions. Macroautophagy appears to rapidly

respond to starvation (1, 6-8) and degrades 30-40% of all rat liver cytosolic

proteins during the first 16-24 h of starvation, but obviously it can not continue for

longer times degrading proteins at such a high rate (11). Thus, the chaperone-

mediated autophagic pathway is primarily activated at later times of starvation,

when macroautophagy declines, and selectively degrades dispensable proteins

containing KFERQ-like sequences (10, 11). The fact that there is DHFR

associated to lysosomes, whose proteases are inhibited by leupeptin, specially

when isolated from rats starved for 88 h (Fig 9), suggests that, in rat liver, DHFR is

transported from the cytosol into lysosomes by chaperone-mediated autophagy.

Methotrexate binds to DHFR and stabilises the tertiary structure of the

protein so that it can not unfold efficiently. We were unable to find any indication

that methotrexate affected lysosomes or the transport of GAPDH (a DHFR-

unrelated protein which does not bind the drug)(Figs. 2-4). We found, in

agreement with others (38, 39), that DHFR is inefficiently transported in vitro into

lysosomes. Therefore, we also used in these experiments a fusion protein of

DHFR with the N-terminal presequence of the precursor polypeptide of the subunit


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9 of the ATP synthase of Neurospora crassa (Su9-DHFR), which is much more

efficiently incorporated (see Table 1). Although this protein does not exist in vivo, it

turned out to be a useful model for the investigation of chaperone-mediated

autophagy. Figs. 10 and 11 and other experiments (see Results) show that the rat

liver lysosomal uptake of this protein also occurs by chaperone-mediated

autophagy. The reason why this fusion protein is incorporated into lysosomes with

such high efficiency is at present unknown. Mouse DHFR contains 2 KFERQ-

related sequences. One is KDRIN (beginning at amino acid 69), if Q can be

replaced by N, and the other is RLIEQ (beginning at amino acid 98). The precursor

polypeptide of the subunit 9 of the ATP synthase of N. crassa contains two

additonal motifs (KRTIQ and LKRTQ, starting at amino acids 33 and 45 in the

sequence) which could be also considered KFERQ-like sequences if T is

phosphorylated. Therefore, it is possible that these two additional KFERQ-like

motifs have a synergistic effect on the lysosomal uptake of DHFR, either by itself

or simply because in the fusion protein the KFERQ-like motifs present in the

DHFR molecule are more accessible to the lysosomal transport machinery. Since

the presequence is in an unfolded state, another explanation is that partially

unfolded proteins are imported with higher efficiencies compared to tightly folded

proteins. In any case, it is clear that addition of the precursor polypeptide

dramatically increases the lysosomal uptake of DHFR (Table 1).

We found that the transport of DHFR and of the DHFR fusion protein into

rat liver lysosomes in vitro was strongly inhibited by addition of methotrexate (Figs.

1B and C, 5B and Table 1). We also observed that inhibition of the uptake of

DHFR was directly related to the methotrexate-induced folding of DHFR (as

assessed by analyses of proteolytic susceptibility)(Figs. 1A and 5A). Moreover,

washing of the drug or addition of dihydrofolate, which competes with

methotrexate for DHFR binding and has a lower affinity of binding to DHFR,

increased the uptake efficiency of methotrexate-treated DHFR (Figs. 6 and 8,

respectively). These observations may probably be explained if both treatments

allow some degree of hsc73-induced unfolding of DHFR. Therefore, DHFR and

probably also other cytosolic proteins are transported into the lysosomes through


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their membranes in an unfolded conformation. According to this model of

transport, and assuming that import of the protein starts at its N-terminal portion

(17), when the fusion protein treated with methotrexate interacts with the

lysosomal membrane, a membrane-spanning translocation intermediate could

accumulate, as the unfolded region crosses the bilayer but the folded moiety gets

"stuck". Since, in the absence of chymostatin, the lysosomal proteases are active,

they should destroy the unfolded region which has entered the lysosome.

Therefore, it may appear surprising that when Su9-DHFR treated with

methotrexate is incubated with lysosomes without chymostatin (i.e. which have

active the internal cathepsins, Fig. 5B, lane 6) no lower molecular mass band is

observed (as it occurs in the incubations with elastase, Fig. 5A, lanes 6-9 and 7A,

lanes 2-9, and with lysosomal cathepsins, data not shown). However, it could be

possible that cutting of this part of the molecule releases the fusion protein from

its binding to the membrane and, in fact, we found, in the supernatants of these

incubations with Su9-DHFR, a lower molecular mass band corresponding to the

DHFR size. Another, or an additional, possibility is that the unfolded portion of the

protein enters initially into a region of the lysosome which is protected from

lysosomal cathepsins. In this regard, it is noteworthy that in most lysosomes of

the dense type, it is possible to detect a narrow electron-lucent rim or band of

clear lysosomal lumen beneath the limiting lysosomal membrane which

separates the inner surface of the lysosomal membrane from the more dense

lysosomal core (see Fig. 3, inset). This area, which has been called a lysosomal

"halo" (47, 48), is believed to correspond to the sugar rich area of lysosomal

proteins on the lysosomal membrane which protects the lysosomal membrane

from the attack by their own hydrolases. Since the width of these area is about 30

nm, it may be possible that the unfolded part of the fusion protein (which is 69

amino acids long) were retained in this part of the lysosomes and, in this way, it

were not accessible to lysosomal proteases. Thus, assuming a size of 0.5 nm per

amino acid, the total length of the N-terminal presequence in its extended

conformation will be about 35 nm, which corresponds approximately to the size of

the lysosomal "halo". If, by analogy with the role in protein transport of other


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intraorganellar heat shock proteins (22, 49), intralysosomal hsc73 (11, 13, 14)

also acts at the trans side of the lysosomal membrane to provide the driving force

to pull the transported protein emerging in the lysosomal lumen, this protective

"halo" may also explain why intralysosomal hsc73, which is susceptible to broken

lysosomes, is not immediately degraded within lysosomes by their cathepsins.

These possibilities merit further investigation, since a translocation intermediate

may be quite useful to identify other componentes of the putative lysosomal

machinery for protein translocation, by procedures similar to those employed with

mitochondria (50, 51).

In summary, DHFR and a DHFR fusion protein can be taken up by

lysosomes, and methotrexate strongly inhibits the uptake. This suggests, as is the

case for the import of proteins into the endoplasmic reticulum and mitochondria,

that DHFR enters lysosomes mainly in an unfolded state. This mechanism is

different from nonclassical protein-transport pathways (42), such as in

peroxisomes, and may explain our earlier unpublished observations that colloidal

gold particles, even when bound to GAPDH, are not efficiently imported in vitro

into isolated rat liver lysosomes. Since binding to the lysosomal membrane is not

affected by methotrexate, it appears that binding to lysosomes is independent of

the folding state of the protein, but that import requires unfolding. In this regard, it

has been shown (52), at least in mitochondria, that certain precursor proteins,

which assume their native forms before mitochondrial import, are unfolded during

translocation. Since hsc73 has been found both in the lysosomal lumen but also

associated to the cytosolic face of the lysosomal membrane (11, 13, 14), it may

be possible that this latter hsc73 fraction produces the required unfolding of the

proteins which are transported into lysosomes for degradation. In addition, DHFR

+ methotrexate can be also incorporated into lysosomes, albeit with low

efficiencies (about 20% of the import without methotrexate, according to the data

in Table 1). Therefore, it appears that either the transport system is not completely

strict in requiring an unfolded protein (as may also occur with other cell

organelles) or that, as previously suggested (37), two different lysosomal uptake

mechanisms coexist in the cell-free assay of lysosomal degradation: a direct


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transfer through the lysosomal membrane corresponding to chaperone-mediated

autophagy and a less effective uptake, under the in vitro conditions used here,

which does not require protein unfolding (most probably microautophagy).


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REFERENCES.

1. Knecht, E., Martín de Llano, J. J., Andreu, E. J., and Moreno Miralles, I.

(1998) Adv. Cell Mol. Biol. 27, 201-234

2. Coux, O., Tanaka, K., and Goldberg, A. L. (1996) Annu. Rev. Biochem. 65,

801-847

3. Mellman, I. (1996) Annu. Rev. Cell Develop. 12, 575-626

4. Lenk, S. E., Fischer, D. L., and Dunn Jr., W. A. (1991) Eur. J. Cell Biol. 56,

201-209

5. Noda, T., and Farquhar, M. G. (1992) J. Cell Biol. 119, 85-97

6. Seglen, P. O., and Bohley, P. (1992) Experientia 48, 158-172

7. Mortimore, G. E., and Kadowaki, M. (1994) in Cellular Proteolytic Systems

(Ciechanover, A. J., and Schwartz, A. L., eds.), pp. 65-87, Wiley-Liss, Inc.,

New York

8. Dunn, W. A., Jr. (1994) Trends Cell Biol. 4, 139-143

9. Dice, J. F. (1990) Trends Biochem. Sci. 15, 305-309

10. Wing, S. S., Chiang, H.-L., Goldberg, A. L., and Dice, J. F. (1991) Biochem. J.

275, 165-169

11. Cuervo, A. M., Knecht, E., Terlecky, S. R., and Dice, J. F. (1995) Am. J. Cell.

Physiol. 269, C1200-C1208

12. Chiang, H.-L., Terlecky, S. R., Plant, C. P., and Dice, J. F. (1989) Science

246, 382-385

13. Agarraberes, F. A., Terlecky, S. R., and Dice, J. F. (1997) J. Cell Biol. 137,

825-834

14. Cuervo, A. M., Dice, J. F., and Knecht, E. (1997) J. Biol. Chem. 272, 5606-

5615

15. Cuervo, A. M., and Dice, J. F. (1996) Science 273, 501-503

16. Aniento, F., Roche, E., Cuervo, A. M., and Knecht, E. (1993) J. Biol. Chem.

268, 10463-10470


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

27

17. Cuervo, A. M., Terlecky, S. R., Dice, J. F., and Knecht, E. (1994) J. Biol.

Chem. 269, 26374-26380

18. Terlecky, S. R., Chiang, H.-L., Olson, T. S., and Dice, J. F. (1992) J. Biol.

Chem. 267, 9202-9209

19. Horst, M., Knecht, E., and Schu, P. V. (1999) Mol. Biol. Cell 10, 2879-2889

20. Baba, M., Osumi, M., Scott, S. V., Klionsky, D. J., and Ohsumi, Y. (1997) J.

Cell Biol. 139, 1687-1695

21. Chiang, M. C., and Chiang, H.-L. (1998) J. Cell Biol. 140, 1347-1356

22. Schatz, G., and Dobberstein, B. (1996) Science 271, 1519-1526

23. Voos, M., Martin, H., Krimmer, T., and Pfanner, N. (1999) Biochim. Biophys.

Acta 1422, 235-254

24. Chen, X., and Schnell, D. J. (1999) Trends Cell Biol. 9, 222-227

25. Subramani, S. (1998) Physiol. Rev. 78, 171-188

26. Dalbey, R. E., and Robinson, C. (1999) Trends Biochem. Sci. 24, 17-22

27. Eilers, M., and Schatz, G. (1986) Nature 322, 228-232

28. Pfanner, N., Müller, H. K., Harmey, M. A., and Neupert, E. (1987) EMBO J. 6,

3449-3454

29. Wattiaux, R., Wattiaux De Conninck, S., Ronveaux-Dupal, M. F., and Dubois,

F. (1978) J. Cell. Biol. 78, 349-368

30. Martín de Llano, J. J., Andreu, E. J., and Knecht, E. (1996) Anal. Biochem.

243, 210-217

31. Aniento, F., Roche, E., and Knecht, E. (1997) Electrophoresis 18, 2638-2644

32. Bennett, J. P. (1982) in Techniques in the Life Sciences, Biochemistry

(Hesketh, T. R., Kornberg, H. L., Metcalfe, J. C., Northcote, D. H., Pogson, C.

X., and Tipton, K. F., eds.), Vol. B4/1, Section B-408, pp. 15-22, Elsevier

Biomedical, Amsterdam

33. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol.

Chem. 193, 265-275

34. Welch, F. J., and Feramisco, J. R. (1985) Mol. Cell Biol. 5, 1229-1237


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

28

35. Laemmli, U. K. (1970) Nature 227, 680-685

36. Robards, A. W., and Wilson, A. J. (eds.)(1993) Procedures in Electron

Microscopy, Section 5, pp. 0.1-4.26, John Wiley & Sons, New York

37. Aniento, F. Papavassiliou, A. G., Knecht, E., and Roche, E. (1996) FEBS Lett.

390, 47-52

38. Chiang, H.-L., and Dice, J. F. (1988) J. Biol. Chem. 263, 6797-6805

39. Terlecky, S. R., and Dice, J. F. (1993). J. Biol. Chem. 268, 23490-23495

40. Cuervo, A. M., and Dice, J. F. (1998) J. Mol. Med. 76, 6-12

41. Blobel, G. (1995) Cold Spring Harb. Symp. Quant. Biol. 60, 1-10

42. Teter, S. A., and Klionsky, D. J. (1999) Trends Cell Biol. 9, 428-431

43. Klionsky, D. J., and Ohsumi, Y. (1999) Annu. Rev. Cell Dev. Biol. 15, 1-32

44. Wilcox, D., and Mason, R. W. (1992) Biochem. J. 285, 495-502

45. Kominami, E., Hashida, S., Khairallah, E. A., and Katunuma, N (1983) J. Biol.

Chem. 258, 6093-6100

46. Ueno, T., Ishidoh, K., Mineki, R., Tanida, I., Murayama, K., Kadowaki, M., and

Kominami, E. (1999) J. Biol. Chem. 274, 15222-15229

47. Holtzmann, E. (1976). Lysosomes: A Survey. Springer Verlag, Wien

48. Daems, W. T., Wisse, E., and Brederoo, P. (1972) in Lysosomes: A

Laboratory Handbook (Dingle, J. T., ed.), pp. 156-199, North Holland

Publishing Company, Amsterdam

49. Neupert, W. (1997) Annu. Rev. Biochem. 66, 863-917

50. Vestweber, D., Brunner, J., Baker, A., and Schatz, G. (1989) Nature 341, 205-

209

51. Horst, M., Hilfiker-Rothenfluh, S., Oppliger, W., and Schatz, G. (1995) EMBO

J. 14, 2293-2297

52. Huang, S., Ratliff, K. S., Schwartz, M. P., Spenner, J. M., and Matouschek, A.

(1999) Nature Struct. Biol. 6, 1132-1138


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FOOTNOTES.

Abbreviations used.1The abbreviations used are: ER, endoplasmic reticulum; hsc73, heat shock

cognate protein of 73 kDa; RNase A, ribonuclease A; RNase S-protein, amino

acids 21-124 of RNase A; DHFR, dihydrofolate reductase; MOPS, 3-[N-

morpholino] propanesulfonic acid; Su9-DHFR, a fusion protein between the

presequence and three amino acid residues of the mature part of subunit 9 of the

F0-ATP synthase of Neurospora crassa and the mouse full length coding region of

DHFR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Acknowledgements.

We are very grateful to J. F. Dice and A. M. Cuervo (Tufts University, Boston) and

M. E. Armengod for carefully reading the manuscript and valuable advice. We also

thank A. Montaner and D. Cerveró for technical assistance.


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FIGURE LEGENDS.

Fig. 1. Methotrexate protects DHFR from proteolysis by elastase (A) and inhibits

its uptake by freshly isolated lysosomes (B,C). A: Effect of methotrexate on the

proteolysis by elastase (1%, w/w) of in vitro synthesised DHFR. Lane 1:

unincubated DHFR; lanes 2-5: DHFR + elastase incubated at 37 ºC for 0, 15, 30

and 60 min; lanes 6-9: DHFR + elastase + methotrexate (250 nM) incubated as in

lanes 2-5, respectively. B and C: Uptake of in vitro synthesised DHFR by freshly

isolated rat liver lysosomes. Aliquots of an in vitro synthesis of DHFR (10 µl of

total synthesis), non-treated (B, - Methotrexate) or treated (C, + Methotrexate) with

methotrexate (250 nM), were incubated (lanes 2-7) in the cell-free assay with

freshly isolated lysosomes (60 µg protein), with (lanes 2-4) or without (lanes 5-7)

30 µM chymostatin, for 10 min at 30 ºC in medium S (see Experimental

Procedures). At the end of the incubation, samples were centrifuged and the

sediments were resuspended in ice-cold 0.3 M sucrose/10 mM MOPS buffer (pH

7.2) and treated (lanes 3, 4, 6, 7) or not (lanes 2, 5) for 10 min at 0 ºC with

proteinase K, in the presence (lanes 4, 7) or not (lanes 3, 6) of Triton X-100, as

described in Experimental Procedures. Then, lysosomes were centrifuged and the

pellets were subjected to SDS-PAGE and fluorography. Lane 1 corresponds to 3%

of the synthesis, which was incubated with the lysosomes. In the presence of

inhibitors of lysosomal proteolysis (lanes 2-4), but not in its absence (lanes 5-7),

DHFR untreated with methotrexate (B), is in part inside lysosomes and, therefore,

protected against proteinase K (lane 3), unless Triton X-100 (lane 4) is added. The

protein associated with lysosomes in the presence of chymostatin (lane 2) roughly

corresponds to the sum of lane 3 (internalised DHFR) and lane 5 (protein

adsorbed to the external surface of lysosomes). Without chymostatin (lanes 5-7),

DHFR associated to lysosomes remains outside, bound to the lysosomal

membrane and, thus, sensitive to exogenously added proteinase K (lane 6).

According to these same criteria, DHFR treated with methotrexate (C) is not

transported and remains outside the lysosomes (compare lanes 3 and 6 in B and


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C). The positions of molecular-mass markers and their size in kDa are indicated

on the left.

Fig. 2. A: Typical ultrastructure of freshly isolated lysosomes from rat liver

incubated for 10 min at 30 ºC in the presence of 250 nM methotrexate. Bar = 0.5

µm. The inset shows an area of this preparation at higher magnification. Note the

clear "halo" (see Discussion) beneath the lysosomal membrane (arrow) which is

visible in most lysosomes of the fraction. Bar = 0.25 µm. No differences were

noticed in the electron microscopic appearance of lysosomes when compared

with an unincubated lysosomal preparation (B). Bar = 0.5 µm.

Fig. 3. Methotrexate plus DHFR neither affects the lysosomal integrity nor the

proteolytic activity of freshly isolated lysosomes. 3H-labelled cytosolic proteins

from CHO cells (about 100,000 cpm), prepared as described in Experimental

procedures, and 10 µl of the reticulocyte lysate transcription-translation mixture,

with (closed symbols) or without (open symbols) DHFR plus 250 nM

methotrexate, were incubated in the cell-free assay under standard conditions

with (n—n, o---o) or without (l—l, m---m) freshly isolated lysosomes (60 µg of

protein). At the indicated times, aliquots were taken to measure proteolysis of the

CHO cells soluble proteins as described in Experimental Procedures. To evaluate

the leakage of proteolytic activity into the medium (lysosomal integrity), lysosomes

were incubated in parallel as above but without the 3H-labelled soluble proteins. At

5, 10, 20, 30 and 40 min, lysosomes were removed by centrifugation,

supernatants were incubated as above with 3H-labelled soluble proteins for the

indicated times and proteolysis was measured (s—s, ∆---∆). The figure shows a

typical experiment. Similar results were obtained with two different preparations.

Fig. 4. A and B: Methotrexate does not affect the transport and uptake of in vitro

synthesised GAPDH by freshly isolated rat liver lysosomes. The experiments were

carried out as in Fig. 1B (Fig. 4A) and 1C (Fig. 4B), except that GAPDH cDNA was


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used instead of DHFR cDNA. Lane 7 corresponds to 6% of the synthesis which

was incubated with the lysosomes. The positions of molecular-mass markers and

their size in kDa are indicated on the left. The histogram (C) below shows the

phosphorimager analysis of the fluorographies: uptake + binding (lane 1), binding

(lane 4), uptake (lane 2).

Fig. 5. A: Effect of methotrexate on the proteolysis of in vitro synthesised Su9-

DHFR by elastase (1%, w/w). Lane 1: unincubated Su9-DHFR; lanes 2-5: Su9-

DHFR + elastase incubated at 37 ºC for 15, 30, 45 and 60 min; lanes 6-9: Su9-

DHFR + elastase + methotrexate incubated, respectively, for the same time

periods as in lanes 2-5. B: Su9-DHFR is efficiently taken up by lysosomes and

methotrexate abolishes this uptake. A cDNA encoding Su9-DHFR was in vitro

transcribed and translated in a rabbit reticulocyte system. Lysosomes (60 µg),

prepared as described in Experimental Procedures, and treated (+) or not (-) with

an inhibitor of lysosomal cathepsins (chymostatin) were incubated in medium S

with Su9-DHFR (10 µl of the reticulocyte lysate transcription-translation mixture)

for 10 min at 30 ºC in the presence (+) or absence (-) of 250 nM methotrexate.

After incubation, the lysosomes were pelleted by centrifugation, washed in 0.3 M

sucrose/10 mM MOPS buffer (pH 7.2), and analysed by SDS-PAGE and

fluorography. Lysosomal uptake can be calculated from the difference in the

values measured for the Su9-DHFR radioactivity associated to the lysosomal

pellets treated (uptake + binding, lanes 3 and 5) or not (binding, lanes 4 and 6)

with chymostatin. Lanes 1 and 2 contain, respectively, 6 and 3% of the

reticulocyte lysate transcription-translation mixture added to the lysosomes. The

positions of molecular-mass markers and their size in kDa are indicated on the

left.

Fig. 6. A: Partial removal of methotrexate increases the uptake efficiency of

methotrexate-treated DHFR by freshly isolated lysosomes. Lysosomes (60 µg)

were incubated, for 10 min at 30 ºC, in a first standard incubation (Incubation 1,


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see Experimental Procedures) with Su9-DHFR (10 µl of the reticulocyte lysate

transcription-translation mixture) in the presence or not of lysosomal protease

inhibitors (30 µM chymostatin) and methotrexate (250 nM), as indicated.

Afterwards, lysosomes were reisolated at 4 ºC, resuspended in medium S in the

absence (lanes 2-5) or in the presence (lanes 6, 7) of the same concentrations of

methotrexate and chymostatin as indicated, and incubated for 10 further minutes

at 30 ºC (Incubation 2). Then, lysosomes were pelleted by centrifugation, washed

in 0.3 M sucrose/10 mM MOPS buffer (pH 7.2), and analysed by SDS-PAGE and

fluorography. Lane 1 contains 6% of the reticulocyte lysate transcription-

translation mixture added to the lysosomes. The positions of molecular-mass

markers and their size in kDa are indicated on the left. The histogram below (B)

shows the phosphorimager analysis of the fluorography. Lysosomal uptake was

calculated as in Fig. 5. The presence (+) or absence (-) of methotrexate (mtx) in

the first and second incubations (separated by a slash) is indicated under

brackets.

Fig. 7. A: Effect of methotrexate (250 nM) and dihydrofolate (250 nM) on the

proteolysis of in vitro synthesised Su9-DHFR by elastase (1%, w/w). Incubations

were carried out at 37 ºC for 15 and 60 min. Lane 1: unincubated Su9-DHFR;

lanes 2-9: Su9-DHFR incubated with elastase for 15 and 60 min, respectively:

without additions (lanes 2, 3) or in the presence of 250 nM dihydrofolate (lanes 4,

5), 250 nM methotrexate (lanes 6, 7) or both together (lanes 8, 9). The positions of

molecular-mass markers and their size in kDa are indicated on the left. The

histogram below (B) shows the phosphorimager analysis of the fluorography. The

presence (+) or absence (-) of methotrexate (mtx) and dihydrofolate (dhf) in the

incubations is indicated.

Fig. 8. Addition of dihydrofolate without (A) or with (C) methotrexate affects the

uptake of in vitro synthesised Su9-DHFR by freshly isolated lysosomes. 5 µl

aliquots of an in vitro synthesis of Su9-DHFR were incubated, in the presence (C)


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or not (A) of methotrexate and without (lanes 2, 3) or with (lanes 3-7) the indicated

concentrations of dihydrofolate, with freshly isolated lysosomes (60 µg protein),

treated (lanes 2, 4, 6) or not (lanes 3, 5, 7) with chymostatin, under standard

conditions. Lane 1 corresponds to 12% of the synthesis which was incubated with

the lysosomes. The positions of molecular-mass markers and their size in kDa are

indicated on the left. The histograms on the right (B, - Methotrexate, and D, +

Methotrexate) show the phosphorimager analysis of the fluorographies on the left

(A and C, respectively). Lysosomal uptake was calculated as in Fig. 5. The

presence (+) or absence (-) of dihydrofolate (dhf) and its concentration when

added (under brackets) are shown.

Fig. 9. Immunolocalisation of DHFR in rat liver fractions. Rats starved for 88 h

were treated or not with leupeptin and liver fractions were prepared as described

under Experimental Procedures. Proteins (50 µg for cytosol and 100 µg for

mitochondria and lysosomes) were separated by SDS-PAGE and stained with

Coomassie Blue R-250 (A) or immunoblotted with anti-DHFR (B and C). A and B:

Cytosol (CYT, lanes 1 and 4), mitochondria (MIT, lanes 2 and 5) and lysosomes

(LYS, lanes 3 and 6) from rats treated (+leupeptin, lanes 4-6) or not (-leupeptin,

lanes 1-3) with leupeptin. C: Cytosolic (lanes 1-3) and lysosomal (lanes 4-6)

fractions isolated from rats treated with leupeptin were incubated for 15 min at 0

ºC without (lanes 1 and 4) or with (lanes 2, 3, 5 and 6) proteinase K (10%, w/w)

and in the presence (lanes 3 and 6) or in the absence (lanes 2 and 5) of 1% Triton

X-100. The positions of molecular-mass markers and their size in kDa are

indicated on the left.

Fig. 10. Effect of ATP/Mg2+ and hsc73 on the uptake of Su9-DHFR by two different

lysosomal populations. A: Su9-DHFR was incubated, as described under

Experimental Procedures, with freshly isolated HSC- (lanes 1-3) and HSC+ (lanes

4-6) lysosomes (i.e. not containing or containing lysosomal hsc73, see

Experimental Procedures), without (lanes 1, 4) or with an ATP-regenerating


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system (ATP) plus hsc73 (lanes 2, 5) or with an ATP-regenerating system alone

(lanes 3, 6). After incubation, samples were treated with proteinase K as

described in Experimental Procedures, centrifuged and pellets and supernatants

were subjected to SDS-PAGE and fluorography. Only lysosomal pellets are

shown; Su9-DHFR in supernatants was totally digested by added proteinase K

(data not shown). The positions of molecular-mass markers and their size in kDa

are indicated on the left. The histogram below (B) shows the mean values of the

phosphorimager analysis of three different fluorographies as in A.

Fig. 11. Effect of RNase A (with KFERQ sequence) and RNase S-protein (without

KFERQ sequence) on the uptake of Su9-DHFR by lysosomes. Lysosomes (60 µg

of protein) treated with 30 µM chymostatin were incubated at 30ºC for 10 min

under standard conditions with an in vitro synthesis of Su9-DHFR without (lane 1)

or with increasing amounts of RNase A (A) and RNase S-protein (B) as labelled

on the figure (lanes 2-5). At the end of the incubation, samples were centrifuged,

the sediments were treated with proteinase K as described in Experimental

Procedures and subjected to SDS-PAGE and fluorography. The positions of

molecular-mass markers and their size in kDa are indicated on the left.


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Table 1

Quantification of the binding and uptake of DHFR and Su9-DHFR, treated or not

with methotrexate, by freshly isolated lysosomes.

Binding + Binding (%) uptake (%) Uptake (%)

DHFR 1.2 ± 0.5 2.6 ± 0.8 1.4 ± 0.3

DHFR + methotrexate 1.1 ± 0.3 1.4 ± 0.4 0.3 ± 0.1

Su9-DHFR 3.4 ± 0.7 14.4 ± 3.1 11.0 ± 2.7

Su9-DHFR+ methotrexate 3.9 ± 0.7 6.1 ± 1.0 2.2 ± 0.4

Quantification, using phosphorimager analysis, of the autoradiograms from four

different experiments as in Fig. 5 (DHFR, two upper lines in the table) and in Fig.

6B (Su9-DHFR, two lower lines in the table). The protein associated to the

lysosomes in the absence (binding, Fig. 5 and 6B, lanes 4 and 6, which

represents the protein adsorbed to the external surface of the lysosomes) or in the

presence (binding + uptake, Fig. 5 and 6B, lanes 3 and 5, which represents

surface bound plus internalised protein) of chymostatin are shown in columns 1

and 2 of the table, respectively. Uptake (column 3 of the table) was calculated

from the difference in the values measured for columns 2 and 1. Values are

expressed as a percentage of the total protein added to the assay. Differences in

uptake, with and without methotrexate, were significant at p < 0.005.


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Natalia Salvador, Carmen Aguado, Martin Horst and Erwin Knechtdepends on its Folding State

Import of a Cytosolic Protein into Lysosomes by Chaperone-Mediated Autophagy

published online June 20, 2000J. Biol. Chem.

10.1074/jbc.M001394200Access the most updated version of this article at doi:

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