FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4256376099> ?p ?o ?g. }

• W4256376099 endingPage "27456" @default.
• W4256376099 startingPage "27447" @default.
• W4256376099 abstract "We have analyzed the folding state of cytosolic proteins imported in vitro into lysosomes, using an approach originally developed by Eilers and Schatz, (Eilers, M., and Schatz, G. (1986) Nature 322, 228–232) to investigate protein import into mitochondria. The susceptibility toward proteases of mouse dihydrofolate reductase (DHFR), synthesized 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 synthesized 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 fromNeurospora 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. We have analyzed the folding state of cytosolic proteins imported in vitro into lysosomes, using an approach originally developed by Eilers and Schatz, (Eilers, M., and Schatz, G. (1986) Nature 322, 228–232) to investigate protein import into mitochondria. The susceptibility toward proteases of mouse dihydrofolate reductase (DHFR), synthesized 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 synthesized 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 fromNeurospora 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. endoplasmic reticulum heat shock cognate protein of 73 kDa ribonuclease A amino acids 21–124 of RNase A dihydrofolate reductase 3-(N-morpholino)propanesulfonic acid a fusion protein between the presequence and 3 amino acid residues of the mature part of subunit 9 of the F0-ATP synthase of N. crassa and the mouse full-length coding region of DHFR glyceraldehyde-3-phosphate dehydrogenase polyacrylamide gel electophoresis Proteins are continuously being degraded by both lysosomal (1Knecht E. Martı́n de Llano J.J. Andreu E.J. Moreno Miralles I. Adv. Cell Mol. Biol. 1998; 27: 201-234Crossref Scopus (10) Google Scholar) and non-lysosomal (2Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2232) Google Scholar) proteases. Lysosomes, which are found in almost all eukaryotic cells, participate in intracellular protein degradation by various mechanisms (1Knecht E. Martı́n de Llano J.J. Andreu E.J. Moreno Miralles I. Adv. Cell Mol. Biol. 1998; 27: 201-234Crossref Scopus (10) Google Scholar): endocytosis, crinophagy, direct conversion of endoplasmic reticulum (ER)1cisternae 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 recognized by specific receptors or simply trapped by nonspecific uptake (3Mellman I. Annu. Rev. Cell Dev. 1996; 12: 575-626Crossref PubMed Scopus (1336) Google Scholar). This is also the degradative pathway followed by plasma membrane proteins which, unlike the low density lipoprotein or the transferrin receptors, do not recycle back to the plasma membrane for reuse. By crinophagy (4Lenk S.E. Fischer D.L. Dunn Jr., W.A. Eur. J. Cell Biol. 1991; 56: 201-209PubMed Google Scholar), 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 (5Noda T. Farquhar M.G. J. Cell Biol. 1992; 119: 85-97Crossref PubMed Scopus (53) Google Scholar). In macroautophagy, or classical autophagy, large areas of cytoplasm, typically including whole organelles, are sequestered by a segregating structure and degraded by lysosomes (6Seglen P.O. Bohley P. Experientia (Basel). 1992; 48: 158-172Crossref PubMed Scopus (368) Google Scholar, 7Mortimore G.E. Kadowaki M. Ciechanover A.J. Schwartz A.L. Cellular Proteolytic Systems. Wiley-Liss, Inc., New York1994: 65-87Google Scholar). 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 internalized into the lysosomal matrix by various modifications of the lysosomal membrane which produce intralysosomal vesicles (8Dunn Jr., W.A. Trends Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (444) Google Scholar). 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+ (9Dice J.F. Trends Biochem. Sci. 1990; 15: 305-309Abstract Full Text PDF PubMed Scopus (529) Google Scholar). Since its original discovery, the chaperone-mediated autophagic pathway for the uptake and degradation of cytosolic proteins has been found to be also operative in rat liver, especially under long-term starvation (10Wing S.S. Chiang H.-L. Goldberg A.L. Dice J.F. Biochem. J. 1991; 275: 165-169Crossref PubMed Scopus (115) Google Scholar, 11Cuervo A.M. Knecht E. Terlecky S.R. Dice J.F. Am. J. Cell. Physiol. 1995; 269: C1200-C1208Crossref PubMed Google Scholar), and in kidney and heart, but not in brain, testes, and skeletal muscle (10Wing S.S. Chiang H.-L. Goldberg A.L. Dice J.F. Biochem. J. 1991; 275: 165-169Crossref PubMed Scopus (115) Google Scholar). This selective uptake of cytosolic proteins requires KFERQ-like sequences (9Dice J.F. Trends Biochem. Sci. 1990; 15: 305-309Abstract Full Text PDF PubMed Scopus (529) Google Scholar), ATP/Mg2+, and a cytosolic (12Chiang H.-L. Terlecky S.R. Plant C.P. Dice J.F. Science. 1989; 246: 382-385Crossref PubMed Scopus (708) Google Scholar) and an intralysosomal (13Agarraberes F.A. Terlecky S.R. Dice J.F. J. Cell Biol. 1997; 137: 825-834Crossref PubMed Scopus (252) Google Scholar, 14Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) hsc73. In addition, the lysosomal membrane glycoprotein lamp-2a was suggested as a receptor for this pathway (15Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (702) Google Scholar). The pathway has been also, at least partially, reconstituted in vitro with lysosomes from rat liver (16Aniento F. Roche E. Cuervo A.M. Knecht E. J. Biol. Chem. 1993; 268: 10463-10470Abstract Full Text PDF PubMed Google Scholar,17Cuervo A.M. Terlecky S.R. Dice J.F. Knecht E. J. Biol. Chem. 1994; 269: 26374-26380Abstract Full Text PDF PubMed Google Scholar) and from human fibroblasts (12Chiang H.-L. Terlecky S.R. Plant C.P. Dice J.F. Science. 1989; 246: 382-385Crossref PubMed Scopus (708) Google Scholar, 18Terlecky S.R. Chiang H.-L. Olson T.S. Dice J.F. J. Biol. Chem. 1992; 267: 9202-9209Abstract Full Text PDF PubMed Google Scholar), as well as with yeast vacuoles (19Horst M. Knecht E. Schu P.V. Mol. Biol. Cell. 1999; 10: 2879-2889Crossref PubMed Scopus (31) Google Scholar). In this latter case, the relationship of the observed uptake with the well established uptake of cytosolic proteins into vesicular intermediates, which is followed by fusion with the vacuolar membrane (20Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (277) Google Scholar, 21Chiang M.C. Chiang H.-L. J. Cell Biol. 1998; 140: 1347-1356Crossref PubMed Scopus (66) Google Scholar), 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 synthesized on cytosolic ribosomes into other cell compartments, such as mitochondria, chloroplasts, and the various types of microbodies (peroxisomes, glycosomes, hydrogenosomes, and glyoxysomes) (22Schatz G. Dobberstein B. Science. 1996; 271: 1519-1526Crossref PubMed Scopus (920) Google Scholar, 23Voos M. Martin H. Krimmer T. Pfanner N. Biochim. Biophys. Acta. 1999; 1422: 235-254Crossref PubMed Scopus (128) Google Scholar, 24Chen X. Schnell D.J. Trends Cell Biol. 1999; 9: 222-227Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 25Subramani S. Physiol. Rev. 1998; 78: 171-188Crossref PubMed Scopus (284) Google Scholar). 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 Refs. 22Schatz G. Dobberstein B. Science. 1996; 271: 1519-1526Crossref PubMed Scopus (920) Google Scholar, 23Voos M. Martin H. Krimmer T. Pfanner N. Biochim. Biophys. Acta. 1999; 1422: 235-254Crossref PubMed Scopus (128) Google Scholar, 24Chen X. Schnell D.J. Trends Cell Biol. 1999; 9: 222-227Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholarfor 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 Refs. 25Subramani S. Physiol. Rev. 1998; 78: 171-188Crossref PubMed Scopus (284) Google Scholar and 26Dalbey R.E. Robinson C. Trends Biochem. Sci. 1999; 24: 17-22Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar 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 (27Eilers M. Schatz G. Nature. 1986; 322: 228-232Crossref PubMed Scopus (466) Google Scholar), 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 stabilized by complexing it with methotrexate. Here, we have analyzed the effect of methotrexate on the uptake by isolated rat liver lysosomes of the cytosolic enzyme DHFR synthesized in vitroin a coupled transcription-translation system with rabbit reticulocytes. From the obtained results we conclude that DHFR passes through the lysosomal membrane mostly in an unfolded conformation. Metrizamide (grade I), trypsin, elastase, trypsin inhibitor, elastatinal, phenylmethylsulfonyl fluoride, chymostatin, chloroquine, methotrexate, dihydrofolate, Triton X-100, MOPS, OsO4, ATP, creatine phosphate, creatine phosphokinase, ATP-agarose,p-nitrophenol-N-d-acetylglucosaminide, 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 Quı́mica 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 inEscherichia coli. Sodium deoxycholate, uranyl acetate, proteinase K, and sucrose were from Merck (Darmstadt, Germany). Bovine serum albumin (fraction V) was from Roche Molecular Biochemicals (Mannheim, Germany). Acrylamide/Bis 29:1 was from Bio-Rad. 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 Life Science Products Inc. (Boston, MA). Other reagents were of the best analytical quality available. A recombinant pSP65 plasmid containing aBamHI-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 (28Pfanner N. Müller H.K. Harmey M.A. Neupert E. EMBO J. 1987; 6: 3449-3454Crossref PubMed Scopus (77) Google Scholar) 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 vitrotranslation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a recombinant pSP64 plasmid, designed pIC328, containing aPstI-BamHI fragment of about 1400 base pairs, which includes the full-length human GAPDH coding region plus additional 5′- and 3′-non-translated sequences (16Aniento F. Roche E. Cuervo A.M. Knecht E. J. Biol. Chem. 1993; 268: 10463-10470Abstract Full Text PDF PubMed Google Scholar), 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. Incubations were carried out at 37 °C 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 of protein/ml). At the times indicated, portions were taken, 50 μmelastatinal 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). 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 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. (29Wattiaux R. Wattiaux De Conninck S. Ronveaux-Dupal M.F. Dubois F. J. Cell Biol. 1978; 78: 349-368Crossref PubMed Scopus (237) Google Scholar). Rats (200–250 g) were anesthetized with ether and decapitated. 10 g of rat liver were used and homogenate (7 ml of chilled 0.25 msucrose/g liver) was filtered through cheesecloth and centrifuged in a Sepatech Biofuge 28RS (Heraeus Sepatech GmbH, Osterode, Germany) at 4,800 × g for 10 min and the supernatant at 17,000 × 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 × 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 × g for 10 min in a Sorvall centrifuge (rotor SS-34). Mitochondria were obtained from the 57–32.8% interface (16Aniento F. Roche E. Cuervo A.M. Knecht E. J. Biol. Chem. 1993; 268: 10463-10470Abstract Full Text PDF PubMed Google Scholar). All procedures were carried out at 0–4 °C. 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 of 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-acetylglucosaminidase (11Cuervo A.M. Knecht E. Terlecky S.R. Dice J.F. Am. J. Cell. Physiol. 1995; 269: C1200-C1208Crossref PubMed Google Scholar). 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 (14Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). These fractions (referred to hereafter as HSC+ and HSC− lysosomes) contained or did not contain, respectively, hsc73 within the lysosomal lumen (Ref. 14Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar and data not shown). Freshly isolated lysosomes (60 μg of protein), treated or not with chymostatin (see below), were incubated, for 10 min at 30 °C 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 vitrosynthesized proteins (10 μl of the standard synthesis reaction, except where indicated), treated or not with 250 nmmethotrexate. Although lysosomes (11Cuervo A.M. Knecht E. Terlecky S.R. Dice J.F. Am. J. Cell. Physiol. 1995; 269: C1200-C1208Crossref PubMed Google Scholar, 13Agarraberes F.A. Terlecky S.R. Dice J.F. J. Cell Biol. 1997; 137: 825-834Crossref PubMed Scopus (252) Google Scholar, 14Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) 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 °C for 10 min at three times its final concentration (30 μm) and then diluted 3-fold with incubation buffer containing the in vitrosynthesized 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 × g for 5 min at 4 °C, 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/tube) in 0.3 msucrose/MOPS buffer, pH 7.2, of the washed sediments was carried out, with or without 1% Triton X-100, for 10 min at 0 °C. 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 synthesized proteins and the following treatments, lysosomes were preincubated for 5 min at 30 °C with 0.3 m 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 synthesized proteins, were carried out as described (17Cuervo A.M. Terlecky S.R. Dice J.F. Knecht E. J. Biol. Chem. 1994; 269: 26374-26380Abstract Full Text PDF PubMed Google Scholar). Chinese hamster ovary cells were grown as described (30Martı́n de Llano J.J. Andreu E.J. Knecht E. Anal. Biochem. 1996; 243: 210-217Crossref PubMed Scopus (16) Google Scholar). They were metabolically labeled for 48 h with 25 μCi/dish [3H]leucine and a cytosolic fraction was prepared as described previously (31Aniento F. Roche E. Knecht E. Electrophoresis. 1997; 18: 2638-2644Crossref PubMed Scopus (4) Google Scholar). Freshly isolated lysosomes (60 μg of protein) were incubated for different times in medium S with the3H-labeled cytosolic proteins from Chinese hamster ovary cells (10 μg of protein with a specific activity of about 10,000 dpm/μg of 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 of 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 (16Aniento F. Roche E. Cuervo A.M. Knecht E. J. Biol. Chem. 1993; 268: 10463-10470Abstract Full Text PDF PubMed Google Scholar), lysosomes were incubated in parallel as above but without the 3H-labeled soluble proteins. At 5, 10, 20, 30, and 40 min, lysosomes were removed by centrifugation and supernatants were incubated with 3H-labeled 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. 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 (32Bennett J.P. Hesketh T.R. Kornberg H.L. Metcalfe J.C. Northcote D.H. Pogson C.X. Tipton K.F. Techniques in the Life Sciences, Biochemistry. B4/1. Elsevier Biomedical, Amsterdam1982: 15-22Google Scholar), of the Lowry et al. (33Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) method and using bovine serum albumin as the standard. Hsc73 was purified from rat liver cytosol by ATP-agarose affinity chromatography (34Welch F.J. Feramisco J.R. Mol. Cell. Biol. 1985; 5: 1229-1237Crossref PubMed Scopus (260) Google Scholar). Cytosol was the supernatant of three successive centrifugations of rat liver homogenates at 2,500 × g, 10 min, 17,000 × g, 10 min and 155,000 × g, 60 min in a Beckman L5–65 centrifuge, Ti-70.1 rotor. SDS-PAGE was done according to Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar) 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 analyzer and Science Lab 98 Image Gauge version 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 (14Cuervo A.M. Dice J.F. Knecht E. J. Biol. Chem. 1997; 272: 5606-5615Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 16Aniento F. Roche E. Cuervo A.M. Knecht E. J. Biol. Chem. 1993; 268: 10463-10470Abstract Full Text PDF PubMed Google Scholar). Lysosomal fractions, treated or not with methotrexate, were fixed and embedded in Epon for conventional electron microscopy by standard procedures (36Robards, A. W., and Wilson, A. J. (eds) (1993) Procedures in Electron Microscopy, pp. 0.1-4.26, John Wiley & Sons, New YorkGoogle Scholar). All data shown are representative results of at least three separate experiments. To test the effect of methotrexate on the conformation of DHFR, aliquots of in vitro synthesized 35S-labeled DHFR, treated or not with methotrexate (250 nm), were incubated with elastase for increasing time periods (Fig.1 A). 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 mixed35S-labeled DHFR with freshly isolated rat liver lysosomes in an isotonic medium at pH 7.2 (see “Experimental Procedures”) in the absence or presence of chymostatin (to inhibit intralysosomal proteolysis) (Fig. 1 B). This in vitro assay, to monitor the uptake of cytosolic proteins into rat liver lysosomes, has been described in detail (e.g. Refs. 16Aniento F. Roche E. Cuervo A.M. Knecht E. J. Biol. Chem. 1993; 268: 10463-10470Abstract Full Text PDF PubMed Google Scholar and 17Cuervo A.M. Terlecky S.R. Dice J.F. Knecht E. J. Biol. Chem. 1994; 269: 26374-26380Abstract Full Text PDF PubMed Google Scholar). 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 and5), 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. 1 B, 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. 1 C), 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 was almost no difference in the amount of DHFR associated to the washed lysosomal pellets with or without chymostatin (lanes 2 and5), and proteinase K released the lysosomal membrane from the associated DHFR (lanes 3 and 6). Methotrexate neither affected the activity of elastase (as observed by digestion of albumin by elastase in the presence of 250 nmmethotrexate, 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-acetylglucosaminidase 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 ▴ with Δ), nor the proteolytic activity of lysosomes toward an extract of cytosolic proteins (compare ▪ with ■). Moreover, w" @default.
• W4256376099 created "2022-05-12" @default.
• W4256376099 creator A5004922762 @default.
• W4256376099 creator A5013701093 @default.
• W4256376099 creator A5040017248 @default.
• W4256376099 creator A5084809317 @default.
• W4256376099 date "2000-09-01" @default.
• W4256376099 modified "2023-09-30" @default.
• W4256376099 title "Import of a Cytosolic Protein into Lysosomes by Chaperone-mediated Autophagy Depends on Its Folding State" @default.
• W4256376099 cites W1517182987 @default.
• W4256376099 cites W1535996154 @default.
• W4256376099 cites W1537517521 @default.
• W4256376099 cites W1542947565 @default.
• W4256376099 cites W1581683718 @default.
• W4256376099 cites W1602095849 @default.
• W4256376099 cites W1603242608 @default.
• W4256376099 cites W1657935059 @default.
• W4256376099 cites W1665138641 @default.
• W4256376099 cites W1775749144 @default.
• W4256376099 cites W1847698873 @default.
• W4256376099 cites W1859261521 @default.
• W4256376099 cites W1968322176 @default.
• W4256376099 cites W1990956809 @default.
• W4256376099 cites W2000910904 @default.
• W4256376099 cites W2001586974 @default.
• W4256376099 cites W2013944211 @default.
• W4256376099 cites W2021706375 @default.
• W4256376099 cites W2022964430 @default.
• W4256376099 cites W2024538345 @default.
• W4256376099 cites W2025933441 @default.
• W4256376099 cites W2026015971 @default.
• W4256376099 cites W2028858498 @default.
• W4256376099 cites W2040253885 @default.
• W4256376099 cites W2069778339 @default.
• W4256376099 cites W2075846958 @default.
• W4256376099 cites W2084520521 @default.
• W4256376099 cites W2088454724 @default.
• W4256376099 cites W2098561880 @default.
• W4256376099 cites W2099301077 @default.
• W4256376099 cites W2100437915 @default.
• W4256376099 cites W2100837269 @default.
• W4256376099 cites W2115027228 @default.
• W4256376099 cites W2115312048 @default.
• W4256376099 cites W2127781535 @default.
• W4256376099 cites W2134344512 @default.
• W4256376099 cites W2137690550 @default.
• W4256376099 cites W2154834069 @default.
• W4256376099 cites W2163508965 @default.
• W4256376099 cites W2164688332 @default.
• W4256376099 cites W2168857111 @default.
• W4256376099 cites W2178119444 @default.
• W4256376099 cites W2178503716 @default.
• W4256376099 cites W2462855682 @default.
• W4256376099 cites W4239776692 @default.
• W4256376099 doi "https://doi.org/10.1016/s0021-9258(19)61529-2" @default.
• W4256376099 hasPublicationYear "2000" @default.
• W4256376099 type Work @default.
• W4256376099 citedByCount "148" @default.
• W4256376099 countsByYear W42563760992012 @default.
• W4256376099 countsByYear W42563760992013 @default.
• W4256376099 countsByYear W42563760992014 @default.
• W4256376099 countsByYear W42563760992015 @default.
• W4256376099 countsByYear W42563760992016 @default.
• W4256376099 countsByYear W42563760992017 @default.
• W4256376099 countsByYear W42563760992018 @default.
• W4256376099 countsByYear W42563760992019 @default.
• W4256376099 countsByYear W42563760992020 @default.
• W4256376099 countsByYear W42563760992021 @default.
• W4256376099 countsByYear W42563760992022 @default.
• W4256376099 countsByYear W42563760992023 @default.
• W4256376099 crossrefType "journal-article" @default.
• W4256376099 hasAuthorship W4256376099A5004922762 @default.
• W4256376099 hasAuthorship W4256376099A5013701093 @default.
• W4256376099 hasAuthorship W4256376099A5040017248 @default.
• W4256376099 hasAuthorship W4256376099A5084809317 @default.
• W4256376099 hasBestOaLocation W42563760991 @default.
• W4256376099 hasConcept C104317684 @default.
• W4256376099 hasConcept C119599485 @default.
• W4256376099 hasConcept C12554922 @default.
• W4256376099 hasConcept C127413603 @default.
• W4256376099 hasConcept C141109615 @default.
• W4256376099 hasConcept C142724271 @default.
• W4256376099 hasConcept C181199279 @default.
• W4256376099 hasConcept C185592680 @default.
• W4256376099 hasConcept C190283241 @default.
• W4256376099 hasConcept C203522944 @default.
• W4256376099 hasConcept C204328495 @default.
• W4256376099 hasConcept C205260736 @default.
• W4256376099 hasConcept C2775932338 @default.
• W4256376099 hasConcept C2775962898 @default.
• W4256376099 hasConcept C2776545253 @default.
• W4256376099 hasConcept C55493867 @default.
• W4256376099 hasConcept C71924100 @default.
• W4256376099 hasConcept C86803240 @default.
• W4256376099 hasConcept C95444343 @default.
• W4256376099 hasConcept C98539663 @default.
• W4256376099 hasConceptScore W4256376099C104317684 @default.
• W4256376099 hasConceptScore W4256376099C119599485 @default.

• next