The Sackler School of Graduate Biomedical Sciences, Department of Cellular and Molecular Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
* Present address: Howard Hughes Medical Institute, Department of Molecular Genetics, Yale University School of Medicine, New Haven, CT 06510, USA
Author for correspondence (e-mail: jdice01{at}granite.tufts.edu)
Accepted April 6, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Lysosome, Proteolysis, Chaperone, Translocation, Transport
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Certain proteins must also be translocated across lysosomal membranes for their degradation. Proteins can be taken up and degraded by lysosomes following several different pathways (Dunn, 1994; Dice, 2000). One of these lysosomal proteolytic pathways is activated in confluent monolayers of cultured cells by withdrawal of serum growth factors (Auteri et al., 1983; Backer et al., 1983), in certain tissues from intact animals during prolonged starvation (Wing et al., 1991; Cuervo et al., 1995), and in yeast in response to nitrogen deprivation (Horst et al., 1999). In this pathway of proteolysis particular cytosolic proteins are transported into lysosomes by mechanisms similar to protein import for residence in organelles such as mitochondria, the endoplasmic reticulum and chloroplasts (Brodsky, 1998; Bauer et al., 2000; Herrmann and Neupert, 2000). For example, transport of protein substrates into lysosomes requires ATP/MgCl2 (Chiang et al., 1989), the cytosolic constitutively expressed protein of the heat shock family of 70 kDa (Hsc70) (Chiang et al., 1989), and Hsc70 in the lysosomal lumen (lyHsc70) (Agarraberes et al., 1997; Cuervo et al., 1997). Substrate proteins contain a pentapeptide motif related to KFERQ (Dice, 1990), and a receptor for substrate proteins resides in the lysosomal membrane and has been identified as the lysosomal-associated membrane protein type 2a (lamp2a) (Cuervo and Dice, 1996). Increasing the levels of lamp2a in the lysosomal membrane increases the activity of the entire proteolytic pathway (Cuervo and Dice, 1996; Cuervo and Dice, 2000), suggesting that lamp2a levels can be a rate-limiting step for chaperone-mediated autophagy.
Several different substrates of this proteolytic pathway bind to Hsc70 and also to lamp2a (Cuervo and Dice, 1996; Dice, 2000). One substrate, RNase S-peptide, which consists of amino acids 1-20 of bovine pancreatic ribonuclease A (RNase A) (Backer et al., 1983), binds to Hsc70 (Terlecky et al., 1992) but not to lamp2a (A. M. Cuervo and J. F. Dice, unpublished). Nevertheless, RNase S-peptide specifically binds to the lysosomal membrane and blocks the binding and uptake of other protein substrates (Terlecky and Dice, 1993; Cuervo et al., 1994). These results suggest that components of the protein binding and import machinery other than lamp2a are also critical for operation of this proteolytic pathway.
The interactions of cytosolic Hsc70 and protein substrates are mediated by cycles of ATP binding and hydrolysis, with the ADP-bound form of Hsc70 having high affinity for protein substrates (Hightower and Leung, 1997). Several molecular chaperones interact with cytosolic Hsc70 and modulate its ATPase activity and the stability of the Hsc70-polypeptide substrate complex (Frydman and Hohfeld, 1997). The heat-inducible protein of the heat shock family of 70 kDa (Hsp70)-interacting protein (Hip) and the heat shock protein of 40 kDa (Hsp40), a DnaJ homologue, act as chaperone enhancers. Hip stimulates the assembly of Hsc70 with Hsp40 and the polypeptide substrate (Hohfeld et al., 1995), whereas Hsp40 stimulates the ATPase activity of Hsc70 (Suh et al., 1999) leading to increased rates of binding and release of polypeptides. The heat shock protein of 90 kDa (Hsp90) recognizes flexible regions of proteins and prevents the proteins from aggregating (Buchner, 1999). Hsp90-Hsp70 organizing protein (Hop) binds to both Hsp90 and Hsc70 and acts as an adapter between the two molecular chaperones (Pratt and Toft, 1997). Hop may also function as a nucleotide exchanger (Gross and Hessefort, 1996). The Bcl2-associated athanogene 1 protein (BAG-1) uncouples the binding of the protein substrate from the ATPase activity of Hsc70 (Bimston et al., 1998) and may act as a postive (Terada and Mori, 2000) or a negative (Luders et al., 2000; Nollen et al., 2000) regulator of Hsc70, perhaps depending upon the BAG-1 isoform (Luders et al., 2000).
A recent study shows that protein substrates must be unfolded to be translocated across the lysosomal membrane during chaperone-mediated autophagy (Salvador et al., 2000). This unfolding is not required for binding of substrates to the lysosomal membrane, so unfolding must occur at the lysosomal surface (Salvador et al., 2000). We now show that a multi-molecular chaperone complex exists at the lysosomal membrane and is required for the translocation of substrate proteins.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proteins, antibodies and protein assays
Purified Hsp90 and Hsp40 proteins were purchased from Stressgen (Victoria, BC, Canada). Hsc70 was purified from bovine brain cytosol as described (Welch and Feramisco, 1985). The monoclonal antibody (mAb) 13D3 against Hsc70 was a generous gift of Joseph Chandler (Maine Biotechnology Services Inc., Portland, ME). The mAbs against Hsp70 and Hsp90 and rabbit polyclonal antibodies against Hsp40 and against the subunit of the chaperonin containing t-complex polypeptide 1 (CCT
) were purchased from Stressgen. The mAb against Hip used for immunoprecipitation was a gift of David Smith (Mayo Clinic, Scottsdale, AZ), and the anti-Hip antibodies used for western blot analysis and transport blocking assays were purchased from Affinity Bioreagents Inc. (Golden, CO). The human antibody against DNA was obtained from the Center for Disease Control (Atlanta, GA). The mAb against BAG-1 was a gift of Shinichi Takayama and John C. Reed (The Burham Institute, La Jolla, CA). The rabbit antibody against Hop was a gift of Michael Lassle (Massachusetts Institute of Technology, Cambridge, MA). The mAb against the p19 protein from the avian myoblastosis virus (AMV-3C2) was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). The antibody against lamp2a was generated as described (Cuervo and Dice, 1996). Antibodies against RNase A were from Rockland Inc. (Gilbertsville, PA), and those against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Amelia Martinez-Ramon, Instituto de Investigaciones Citologicas, Valencia, Spain. Rabbit serum was purchased from Sigma Chemical Co. (St Louis, MO). Rhodamine-labeled goat anti-rat IgG was purchased from Chemicon (Temecula, CA). All other fluorescence- and horseradish peroxidase-labeled secondary antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Protein determinations were performed using the Lowry assay (Lowry et al., 1951) or the BioRad protein assay reagent (BioRad Laboratories, Hercules, CA).
One- and two-dimensional gel electrophoresis
One dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described (Terlecky and Dice, 1993). Two-dimensional electrophoresis was carried out as described (OFarrell, 1975) with the following modification: samples were boiled for 3 minutes in 0.5% SDS, 10 mM DTT, 10 mM Tris-HCl buffer, pH 6.8, before incubation in isoelectric focusing solubilization buffer to inactivate lysosomal proteases. Isoelectric focusing was carried out as previously described (Agarraberes et al., 1997). For two-dimensional analysis lysosomal membranes were purified as described above, except that they were not washed in 0.5 M NaCl.
Immunoblotting
Proteins were electrotransferred onto Immobilon-P membranes (Millipore Corp., Bedford, MA). Immunoblotting was carried out as previously described (Agarraberes et al., 1997). Antibodies and dilutions were as follows: mouse mAb 13D3 (1:5000), mouse anti-Hsp70 (1:1000), rabbit anti-lamp2a (1:5000), rat anti-Hsp90 (1:5000), rabbit anti-Hsp40 (1:5000), rabbit anti-Hop (1: 3000), mouse anti-Hip (1:5000), mouse anti-BAG-1 (1:300), mouse anti-CCT (1:1000), rabbit anti-GAPDH (1:2000), and rabbit anti-RNase A (1:2000)
Determination of protein stoichiometry
The ratio among Hsc70, Hsp90 and Hsp40 was determined by western blot analysis of serial dilutions of the purified proteins and different concentrations of purified lysosomal membranes. Western blots were developed with chemiluminescense methods (Renaissacence©, NEN-Life Science Products, Boston, MA), and the signals were quantified by densitometry. The lowest dilution of each lysosomal membrane protein presenting a chemiluminescense signal that closely matched the value of one of the dilutions of its corresponding purified protein was used for calculation.
Immunoprecipitations
Beads of concanavalin A (ConA) linked to sepharose (Sigma Chemical Co.) were washed three times in PBS (Terlecky et al., 1992). The mAbs 13D3, anti-Hip and anti-p19 were incubated with ConA sepharose beads for 2 hours at 25°C in PBS, washed three times in PBS, and resuspended in immunoprecipitation (IP) buffer containing 150 mM NaCl, 20 mM Tris-HCl, pH 8.2, 1% Nonidet P-40. Lysosomal membranes were solubilized in IP buffer and centrifuged at 130,000 g for 30 minutes. Supernatants containing solubilized lysosomal membrane proteins were incubated with ConA sepharose-antibody for 2 hours at 4°C, washed twice with IP buffer, and washed once with PBS. SDS-PAGE solubilization buffer (Agarraberes et al., 1997) was added, and samples were processed for electrophoresis.
Immunofluorescence and microscopy
For indirect immunofluorescence studies, cells were handled as previously described (Agarraberes et al., 1997). Briefly, cells were fixed in cold methanol (-20°C) for 1 minute, subjected to triple immunostaining (Agarraberes et al., 1997), and viewed by confocal microscopy (Odyssey XL, Noran Instruments, Middleton, WI). Primary antibodies were used as 1:25 dilutions, and fluorescent-labeled secondary antibodies were used in dilutions according to the manufacturers recommendations. Omission of primary antibodies resulted in no visible fluorescence (data not shown).
In vitro lysosomal import assays, protease protection assays and antibody inhibition of protein transport
Samples were treated as previously described (Terlecky and Dice, 1993). Briefly, purified lysosomes (100 µg protein) were incubated in a final volume of 0.1 ml in an ice bath for 10 minutes in 100 µM chymostatin A (Sigma Chemical Co.) to inhibit digestion of translocated substrate proteins (Cuervo et al., 1994). Samples were then incubated for 20 minutes with no serum or increasing amounts of the following serums: mouse serum containing anti-Hip, nonimmune mouse serum, rabbit serum containing anti-Hsp40, rabbit serum containing anti-Hop, and nonimmune rabbit serum. Samples were incubated for 15 minutes at 37°C in an ATP-regenerating system (Terlecky and Dice, 1993) with either RNase A (Sigma Chemical Co.), GAPDH (Boehringer Mannheim, Indianapolis, IN), or no substrate. Finally, lysosomes were treated with 10 µg of Proteinase K and 1 µM CaCl2 for 20 minutes in an ice bath. The reaction was stopped with 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (Sigma Chemical Co.). Lysis buffer was added, and samples were processed for SDS-PAGE. All reagents were prepared in 0.25 M ultra pure grade sucrose (Sigma Chemical Co.), 5 mM Tris-HCl, pH 7.3.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We analyzed the interaction of lymHsc70 with a protein substrate of this proteolytic pathway, RNase A (Backer et al., 1983; Dice, 2000), using a protease protection assay. Purified lysosomes were incubated either in the presence or absence of ADP/MgCl2 and/or RNase A at 37°C to favor protein binding to lymHsc70 and transport initiation (Fig. 1c). Lysosomes were treated with proteinase K and membranes were purified and analyzed by SDS-PAGE and then immunoblotted with the Hsc70-specific antibody, 13D3. These results indicate that the protein substrate in the presence of ADP/MgCl2 has a protective effect on Hsc70 and that a conformational change in Hsc70 might take place during binding and transport of the protein substrate.
We next explored whether or not lymHsc70 might be in a complex with other chaperones as has been described for cytosolic Hsc70 (Hohfeld et al., 1995; Gross and Hessefort, 1996; Frydman and Hohfeld, 1997; Demand et al., 1998; Stuart et al., 1998; Gross et al., 1999; King et al., 1999). Confluent cultures of cells were serum-deprived for 18 hours. Purified lysosomal membranes were detergent-solubilized and subjected to immunoprecipitation as described in Materials and Methods. Solubilized membranes were incubated with either anti-Hsc70, anti-Hip, or an unrelated antibody to the p19 protein from the avian myoblastosis virus. Western blot analysis of the immunoprecipitates was performed with specific antibodies against Hsp90, Hsc70, Hop, Hip, BAG-1, Hsp40, Hsp70 and CCT (Kubota et al., 1994). Fig. 2 shows the presence on the lysosomal membrane of multiple molecular chaperones and cochaperones known to interact with cytosolic Hsc70 (Hohfeld et al., 1995; Gross and Hessefort, 1996; Frydman and Hohfeld, 1997; Demand et al., 1998; Stuart et al., 1998; Gross et al., 1999; King et al., 1999). Interestingly, anti-Hip antibodies also immunoprecipitate the same set of chaperones/cochaperones, including BAG-1 (Fig. 2). Hip and BAG-1 have overlapping binding sites on the N-terminal domain of Hsc70 and cannot bind to the same molecule of Hsc70 (Demand et al., 1998). Therefore, multiple complexes of lymHsc70 must be present at the lysosomal membrane. Neither Hsp70 nor CCT
was present in the immunoprecipitates even though they are readily detectable in cytosol (Fig. 2) and, to a more variable extent, associated with solubilized lysosomal membranes (data not shown). In addition, immunoprecipitation with an unrelated antibody to p19 failed to immunoprecipitate any of the proteins analyzed (Fig. 2). Finally, we also identified GAPDH, a natural substrate of the pathway, as part of this large complex of chaperones (data not shown). This result indicates that at least some of the chaperone complexes are likely to be functional.
|
These results were in marked contrast to our findings in the lysosomal lumen, where only lyHsc70 was present; although lyHsc70 could be readily detected by immunoblotting, Hsp90, Hop, Hip, BAG-1 and Hsp40 were absent from the lysosomal lumen (data not shown). Hsc70 is known to enter the lysosomal lumen (Agarraberes et al., 1997; Cuervo et al., 1997). Whether the other proteins failed to enter the lysosomes with Hsc70 or were rapidly degraded within the lysosomes after entry is not known.
We fixed serum-deprived IMR-90 human fibroblasts with methanol at -20°C. to remove cytosolic proteins. Previous studies showed that Hsc70 colocalized with the majority (>90%) of vesicular structures containing lysosome-associated membrane protein 1 (Agarraberes et al., 1997), a lysosomal marker (Green et al., 1987). We examined the lysosomal localization of the chaperones and cochaperones by confocal microscopy. Triple immunostaining of fibroblasts was performed using Hsc70 as a lysosomal marker, anti-human DNA antibodies as a nuclear marker, and specific antibodies against the other proteins as shown in Fig. 3. Specific antibodies against the molecular chaperones and cochaperones Hsp90, Hop, BAG-1, Hsp40 and Hip (red signal) largely colocalized with Hsc70 (green signal). Colocalization in the merged images registers as orange/yellow. Quantitation of the merged images after magnification indicated the following degrees of colocalization of Hsc70 and the other chaperones: Hsp90 (97%), Hop (88%), BAG-1 (97%), Hsp40 (95%) and Hip (98%). These results confirm our conclusion that a complex of molecular chaperones exists at the lysosomal membrane.
|
|
We directly assessed the requirement for Hip, Hop and Hsp40 in the process of binding and transport of substrate proteins using antibodies against these proteins. Previous studies showed that antibodies to the cytosolic tail of lamp2a inhibited binding and uptake of substrate proteins (Cuervo and Dice, 1996). In addition, incubation of isolated lysosomes with the antibody to Hsc70 blocked binding and uptake of substrate proteins (A. M. Cuervo and J. F. Dice, unpublished). Purified lysosomes were preincubated with increasing amounts of each specific antibody, and either RNase A or GAPDH was used as substrate. Fig. 5 shows the concentration-dependent inhibitory effect of serum containing antibodies against Hip, Hop and Hsp40 on substrate transport in a cell-free system. Control assays using appropriate nonimmune serum showed no effect on transport of protein substrates. The lower panels in Fig. 5 show the densitometric quantification of these results. These results indicate that Hip, Hop and Hsp40 are required for import of substrate proteins into lysosomes. The mAb against Hsc70 also inhibited translocation of RNase A (data not shown). The antibodies to Hsp90 and BAG-1 did not efficiently immunoprecipitate the native proteins, so they could not be used in this type of analysis. The requirement for multiple molecular chaperones in this lysosomal proteolytic pathway justifies the term chaperone-mediated autophagy to distinguish this pathway from microautophagy and macroautophagy.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Whether or not all lymHsc70 is in such large chaperone complexes remains to be determined. Our immunoprecipitation assays contained solubilized lysosomal membrane proteins in excess, so lack of immunoprecipitation did not necessarily reflect lack of a particular protein in the complex. Our limited stoichiometry results suggest that lymHsc70 is associated with substoichiometric levels of Hsp40 and Hsp90. More complete studies with cytosolic Hsc70 suggest that Hsp40 and Hop are present at one-tenth the amount of Hsc70 (Kosano et al., 1998), whereas Hip (Hohfeld et al., 1995) and BAG-1 (Stuart et al., 1998; Nollen et al., 2000) are approximately equimolar with Hsc70. The amount of Hsp90 in the molecular chaperone complex is highly variable depending on the tissue type and physiological status (Schumacher et al., 1994; Kimmins and MacRae, 2000). Whether or not additional regulators of cytosolic Hsc70 activity such as the C-terminus of Hsc70-interacting protein (Ballinger et al., 1999), Scythe (Kaye et al., 2000; Thress et al., 2001) or Reaper (Thress et al., 2001) are in the lymHsc70 complex remain to be determined
The requirement for rapid and dramatic changes in cellular protein composition during starvation has resulted in a mechanism for identifying protein substrates (the KFERQ motif) and a process of targeting a large amount of proteins to lysosomes for degradation. The involvement of cytosolic Hsc70, lymHsc70 and lamp2a may represent a triple checking mechanism for accurate identification of substrates. These cytosolic proteins are degraded in a membrane-confined compartment rich in hydrolases of rather low specificity. With this system a large number of proteins can be degraded to single amino acids. These amino acids can then be used for synthesis of new proteins or as an energy source.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agarraberes, F., Terlecky, S. and Dice, J. F. (1997). An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation. J. Cell Biol. 137, 825-834.
Auteri, J. S., Okada, A., Bochaki, V. and Dice, J. F. (1983). Regulation of intracellular protein degradation in IMR-90 human diploid fibroblasts. J. Cell. Physiol. 115, 167-174.[Medline]
Backer, J. M., Bourret, L. and Dice, J. F. (1983). Regulation of catabolism of microinjected ribonuclease A requires the amino-terminal 20 amino acids. Proc. Natl. Acad. Sci. USA 80, 2166-2170.[Abstract]
Ballinger, C. A., Connell, P., Wu, Y., Hu, Z., Thompson, L. J., Yin, L. Y. and Patterson, C. (1999). Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol. Cell Biol. 19, 4535-4545.
Bauer, M. F., Hofmann, S., Neupert, W. and Brunner, M. (2000). Protein translocation into mitochondria: the role of TIM complexes. Trends Cell Biol. 10, 25-31.[Medline]
Bernstein, H. D. (2000). The biogenesis and assembly of bacterial membrane proteins. Curr. Opin. Microbiol. 3, 203-209.[Medline]
Bimston, D., Song, J., Winchester, D., Takayama, S., Reed, J. C. and Morimoto, R. I. (1998). BAG-1, a negative regulator of Hsp70 chaperone activity, uncouples nucleotide hydrolysis from substrate release. EMBO J. 17, 6871-6878.
Breyton, C., de Vitry, C. and Popot, J. L. (1994). Membrane association of cytochrome b6f subunits. The Rieske iron-sulfur protein from Chlamydomonas reinhardtii is an extrinsic protein. J. Biol. Chem. 269, 7597-7602.
Brodsky, J. L. (1998). Translocation of proteins across the endoplasmic reticulum membrane. Int. Rev. Cytol. 178, 277-328.[Medline]
Buchner, J. (1999). Hsp90 & Co. - a holding for folding. Trends Biochem. Sci. 24, 136-141.[Medline]
Chiang, H. L., Terlecky, S. R., Plant, C. P. and Dice, J. F. (1989). A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 246, 382-385.[Medline]
Cuervo, A. M. and Dice, J. F. (1996). A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273, 501-503.[Abstract]
Cuervo, A. M. and Dice, J. F. (2000). Unique properties of lamp2a compared to other lamp2 isoforms. J. Cell. Sci. 113, 4441-4450.
Cuervo, A. M., Terlecky, S. R., Dice, J. F. and Knecht, E. (1994). Selective binding and uptake of ribonuclease A and glyceraldehyde-3-phosphate dehydrogenase by isolated rat liver lysosomes. J. Biol. Chem. 269, 26374-26380.
Cuervo, A. M., Knecht, E., Terlecky, S. R. and Dice, J. F. (1995). Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation. Am. J. Physiol. 269, C1200-C1208.
Cuervo, A. M., Dice, J. F. and Knecht, E. (1997). A population of rat liver lysosomes responsible for the selective uptake and degradation of cytosolic proteins. J. Biol. Chem. 272, 5606-5615.
Demand, J., Luders, J. and Hohfeld, J. (1998). The carboxyl-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors. Mol. Cell. Biol. 18, 2023-2028.
Dice, J. F. (1990). Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem. Sci. 15, 305-309.[Medline]
Dice, J. F. (2000). Lysosomal Pathways of Protein Degradation. Austin, TX: Landes Bioscience.
Dunn, W. A. (1994). Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Cell Biol. 4, 139-143.
Frydman, J. and Hohfeld, J. (1997). Chaperones get in touch: the Hip-Hop connection. Trends Biochem. Sci. 22, 87-92.[Medline]
Green, S., Zimmer, K. P., Griffiths, A. and Mellman, I. (1987). Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins. J. Cell Biol. 105, 1227-1240.[Abstract]
Gross, M. and Hessefort, S. (1996). Purification and characterization of a 66-kDa protein from rabbit reticulocyte lysate which promotes the recycling of hsp70. J. Biol. Chem. 271, 16833-16841.
Gross, M., Hessefort, S. and Olin, A. (1999). Purification of a 38-kDa protein from rabbit reticulocyte lysate which promotes protein renaturation by heat shock protein 70 and its identification as delta-aminolevulinate acid dehydratase and as a putative DnaJ protein. J. Biol. Chem. 274, 3125-3134.
Herrmann, J. M. and Neupert, W. (2000). Protein transport into mitochondria. Curr. Opin. Microbiol. 3, 210-214.[Medline]
Hightower, L. and Leung, S. M. (1997). Mammalian Hsc70 and Hsp70. In Guidebook to Molecular Chaperones and Protein-Folding Catalysis (ed. M. J. Gething), pp. 53-58. London: Oxford University Press.
Hohfeld, J., Minami, Y. and Hartl, F.-U. (1995). Hip, a novel cochaperone involved in the eukaryotic hsc70/hsp40 reaction cycle. Cell 83, 589-598.[Medline]
Horst, M., Knecht, E. and Schu, P. V. (1999). Import into and degradation of cytosolic proteins by isolated yeast vacuoles. Mol. Biol. Cell. 10, 2879-2889.
Kaye, F. J., Modi, S., Ivanovska, I., Koonin, E. V., Thress, K., Kubo, A., Kornbluth, S. and Rose, M. D. (2000). A family of ubiquitin-like proteins binds the ATPase domain of Hsp70-like Stch. FEBS Lett. 467, 348-355.[Medline]
Kermorgant, M., Bonnefoy, N. and Dujardin, G. (1997). Oxa1p, which is required for cytochrome c oxidase and ATP synthase complex formation, is embedded in the mitochondrial inner membrane. Curr. Genet. 4, 302-307.
Kimmins, S. and MacRae, T. H. (2000). Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins. Cell Stress Chaperones 5, 76-86.[Medline]
King, C., Eisenberg, E. and Greene, L. E. (1999). Interaction between Hsc70 and DnaJ homologues: relationship between Hsc70 polymerization and ATPase activity. Biochemistry 38, 12452-12459.[Medline]
Kosano, H., Stensgard, B., Charlesworth, M. C., McMahon, N. and Toft, D. (1998). The assembly of progesterone receptor-hsp90 complexes using purified proteins. J. Biol. Chem. 273, 32973-32979.
Kourtz, L. and Ko, K. (1997). The early stage of chloroplast protein import involves Com70. J. Biol. Chem. 272, 2808-2813.
Kubota, H., Hynes, G., Carne, A., Ashworth, A. and Willison, K. (1994). Identification of six Tcp-1-related genes encoding divergent subunits of the Tcp-1-containing chaperonin. Curr. Biol. 4, 89-99.[Medline]
Lin, P., Le-Niculescu, H., Hofmeister, R., McCaffery, J. M., Jin, M., Hennemann, H., McQuistan, T., De Vries, L. and Farquhar, M. G. (1998). The mammalian calcium-binding protein, nucleobindin (CALNUC), is a Golgi resident protein. J. Cell Biol. 141, 1515-1527.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.
Luders, J., Demand, J., Papp, O. and Hohfeld, J. (2000). Distinct isoforms of the cofactor BAG-1 affect Hsc70 chaperone function. J. Biol. Chem. 275, 14817-14823.
Neff, N., Bourret, E., Miao, P. and Dice, J. F. (1981). Degradation of proteins microinjected into IMR-90 human diploid fibroblasts. J. Cell Biol. 91, 184-194.[Abstract]
Nishiyama, K., Fukuda, A., Norita, K. and Tokuda, H. (1999). Membrane deinsertion of SecA underlying proton motive force-dependent stimulation of protein translocation. EMBO J. 18, 1049-1058.
Nollen, E. A., Brunsting, J. F., Song, J., Kapinga, H. H. and Morimoto, R. I. (2000). Bag-1 functions in vivo as a negative regulator of hsp70 chaperone activity. Mol. Cell. Biol. 20, 1083-1088.
OFarrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007-4021.[Abstract]
Ohsumi, Y., Ishikawa, T. and Kato, K. (1983). A rapid and simplified method for the preparation of lysosomal membranes from rat liver. J. Biochem. 93, 547-556.[Abstract]
Otto, J. C. and Smith, W. L. (1996). Photolabeling of prostaglandin endoperoxide H synthase-1 with 3-trifluoro-3-(m-[I-125}iodophenyl)diazirine as a probe of membrane association and the cyclooxygenase active site. J. Biol. Chem. 271, 9906-9910.
Pratt, W. B. and Toft, D. O. (1997). Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endo. Rev. 18, 306-360.
Salvador, N., Aguado, C., Horst, M. and Knecht, E. (2000). Import of a cytosolic protein into lysosomes by chaperone-mediated autophagy depends on its folding state. J. Biol. Chem. 275, 27447-27453.
Schumacher, R. J., Hurst, R., Sullivan, W. P., McMahon, N. J., Toft, D. O. and Matts, R. I. (1994). ATP-dependent chaperoning activity of reticulocyte lysate. J. Biol. Chem. 269, 9493-9499.
Storrie, B. and Madden, E. A. (1990). Isolation of subcellular organelles. Methods Enzymol. 182, 203-225.[Medline]
Stuart, J. K., Myszka, D. G., Joss, L., Mitchell, R. S., McDonald, S. M., Xie, Z., Takayama, S., Reed, J. C. and Ely, K. R. (1998). Characterization of interactions between the anti-apoptotic protein BAG-1 and hsc70 molecular chaperones. J. Biol. Chem. 273, 22506-22514.
Suh, W. C., Lu, C. Z. and Gross, C. A. (1999). Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ. J. Biol. Chem., 30534-30539.
Terada, K. and Mori, M. (2000). Human DnaJ homologs dj2 and dj3 and bag-1 are positive cochaperones of hsc70. J. Biol. Chem. 275, 24728-24734.
Terlecky, S. R. and Dice, J. F. (1993). Polypeptide import and degradation by isolated lysosomes. J. Biol. Chem. 268, 23490-23495.
Terlecky, S. R., Chiang, H. L., Olson, T. S. and Dice, J. F. (1992). Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat shock cognate protein. J. Biol. Chem. 267, 9202-9209.
Teter, S. and Klionsky, D. J. (1999). How to get a folded protein across a membrane. Trends Biochem. Sci. 9, 428-431.
Thress, K., Song, J., Morimoto, R. I. and Kornbluth, S. (2001). Reversible inhibition of Hsp70 chaperone function by Scythe and Reaper. EMBO J. 20, 1033-1041.
Welch, W. J. and Feramisco, J. R. (1985). Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides. Mol. Cell. Biol. 5, 1229-1237.[Medline]
Wiemer, E. A. C., Luers, G. H., Faber, K. N., Wenzel, T., Veenhuis, M. and Subramani, S. (1996). Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. J. Biol. Chem. 271, 18973-18980.
Wing, S. S., Chiang, H. L., Goldberg, A. L. and Dice, J. F. (1991). Proteins containing peptide sequences related to KFERQ are selectively depleted in liver and heart, but not skeletal muscle, of fasted rats. Biochem. J. 275, 165-169.[Medline]
YaDeau, J. T. and Blobel, G. (1989). Solubilization and characterization of yeast signal peptidase. J. Biol. Chem. 264, 2928-2934.