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An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation.

Agarraberes FA, Terlecky SR, Dice JF - J. Cell Biol. (1997)

Bottom Line: The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected.The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3.These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

View Article: PubMed Central - PubMed

Affiliation: Sackler School of Graduate Biomedical Sciences, Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
Previous studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules. Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [3H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

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Stability of endocytosed proteins. Fibroblasts were  incubated overnight in media containing: 35S-labeled cytosolic  proteins from IMR-90 fibroblasts (cytosol), [3H]RNase A, total  [3H]hsc73 (HSC73), or [3H]hsc73 that had been repurified by  ATP-affinity chromatography (HSC73*). Cultures were chased  for 6 h in media containing 10% NCS, and then degradation was  followed by measuring acid-soluble radioactivity appearing in the  medium. Results are the mean for n = 3–6.
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Figure 8: Stability of endocytosed proteins. Fibroblasts were incubated overnight in media containing: 35S-labeled cytosolic proteins from IMR-90 fibroblasts (cytosol), [3H]RNase A, total [3H]hsc73 (HSC73), or [3H]hsc73 that had been repurified by ATP-affinity chromatography (HSC73*). Cultures were chased for 6 h in media containing 10% NCS, and then degradation was followed by measuring acid-soluble radioactivity appearing in the medium. Results are the mean for n = 3–6.

Mentions: The ly-hsc73 appears to be cytosolic hsc73 that enters lysosomes through macroautophagy or other processes and is slightly modified to become more acidic. If ly-hsc73 is hsc73, we reasoned that hsc73 might be particularly resistant to intralysosomal digestion. We measured the degradation rates of RNase A, a mixture of cytosolic proteins, and two different preparations of hsc73 after endocytosis (Fig. 8). RNase A was degraded with a half-life of 47 h, while the half-lives of cytosolic proteins were heterogeneous in the 20–60-h range (data not shown). Radiolabeled hsc73 that was repurified by ATP-affinity chromatography before endocytosis was remarkably stable (t1/2 = 170 h). Degradation of total reductively methylated hsc73, most of which could not bind to ATP, was biphasic with rapid (t1/2 = 5.5 h) and longer-lived (t1/2 = 26 h) components. These results are consistent with most of the [3H]hsc73 being denatured by the reductive methylation and short-lived after endocytosis, while the native [3H]hsc73 is very stable within lysosomes.


An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation.

Agarraberes FA, Terlecky SR, Dice JF - J. Cell Biol. (1997)

Stability of endocytosed proteins. Fibroblasts were  incubated overnight in media containing: 35S-labeled cytosolic  proteins from IMR-90 fibroblasts (cytosol), [3H]RNase A, total  [3H]hsc73 (HSC73), or [3H]hsc73 that had been repurified by  ATP-affinity chromatography (HSC73*). Cultures were chased  for 6 h in media containing 10% NCS, and then degradation was  followed by measuring acid-soluble radioactivity appearing in the  medium. Results are the mean for n = 3–6.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139836&req=5

Figure 8: Stability of endocytosed proteins. Fibroblasts were incubated overnight in media containing: 35S-labeled cytosolic proteins from IMR-90 fibroblasts (cytosol), [3H]RNase A, total [3H]hsc73 (HSC73), or [3H]hsc73 that had been repurified by ATP-affinity chromatography (HSC73*). Cultures were chased for 6 h in media containing 10% NCS, and then degradation was followed by measuring acid-soluble radioactivity appearing in the medium. Results are the mean for n = 3–6.
Mentions: The ly-hsc73 appears to be cytosolic hsc73 that enters lysosomes through macroautophagy or other processes and is slightly modified to become more acidic. If ly-hsc73 is hsc73, we reasoned that hsc73 might be particularly resistant to intralysosomal digestion. We measured the degradation rates of RNase A, a mixture of cytosolic proteins, and two different preparations of hsc73 after endocytosis (Fig. 8). RNase A was degraded with a half-life of 47 h, while the half-lives of cytosolic proteins were heterogeneous in the 20–60-h range (data not shown). Radiolabeled hsc73 that was repurified by ATP-affinity chromatography before endocytosis was remarkably stable (t1/2 = 170 h). Degradation of total reductively methylated hsc73, most of which could not bind to ATP, was biphasic with rapid (t1/2 = 5.5 h) and longer-lived (t1/2 = 26 h) components. These results are consistent with most of the [3H]hsc73 being denatured by the reductive methylation and short-lived after endocytosis, while the native [3H]hsc73 is very stable within lysosomes.

Bottom Line: The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected.The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3.These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

View Article: PubMed Central - PubMed

Affiliation: Sackler School of Graduate Biomedical Sciences, Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
Previous studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules. Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [3H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

Show MeSH
Related in: MedlinePlus