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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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Effect of mAb 13D3 on enhanced degradation of cellular proteins  in response to serum deprivation. Fibroblasts were labeled with [3H]leucine for 2 d, and then incubated overnight in medium containing 10% NCS  and either no addition (NONE), or  mAb 13D3 (13D3), mAb 13D3 preincubated with hsc73 (13D3/HSC73), or  mAb p32 (P32). The cells were chased  with fresh media containing 10% NCS  for 1 h, and then changed to serumsupplemented (solid lines) or serumdeprived (dotted lines) media. Acidsoluble radioactivity in the media was  followed as a measure of proteolysis.  Results shown are the mean for n = 6  (NONE), or n = 4 (13D3, 13D3/ HSC73, and P32). The half-lives (in h)  in the presence and absence of serum,  respectively, are: (A) 80 and 52; (B) 81  and 84; (C) 100 and 74; and (D) 120  and 77.
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Figure 6: Effect of mAb 13D3 on enhanced degradation of cellular proteins in response to serum deprivation. Fibroblasts were labeled with [3H]leucine for 2 d, and then incubated overnight in medium containing 10% NCS and either no addition (NONE), or mAb 13D3 (13D3), mAb 13D3 preincubated with hsc73 (13D3/HSC73), or mAb p32 (P32). The cells were chased with fresh media containing 10% NCS for 1 h, and then changed to serumsupplemented (solid lines) or serumdeprived (dotted lines) media. Acidsoluble radioactivity in the media was followed as a measure of proteolysis. Results shown are the mean for n = 6 (NONE), or n = 4 (13D3, 13D3/ HSC73, and P32). The half-lives (in h) in the presence and absence of serum, respectively, are: (A) 80 and 52; (B) 81 and 84; (C) 100 and 74; and (D) 120 and 77.

Mentions: In contrast, the enhanced protein degradation in response to serum withdrawal (Fig. 6 A) was blocked when the mAb was endocytosed alone (Fig. 6 B). Endocytosis of mAb 13D3 previously neutralized with hsc73 (Fig. 6 C), or the control antibody (P32; Fig. 6 D), had little effect. These results were obtained in two additional experiments except that the slightly slower protein degradation observed for the P32-treated cells (Fig. 6 D) was not a consistent finding.


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

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

Effect of mAb 13D3 on enhanced degradation of cellular proteins  in response to serum deprivation. Fibroblasts were labeled with [3H]leucine for 2 d, and then incubated overnight in medium containing 10% NCS  and either no addition (NONE), or  mAb 13D3 (13D3), mAb 13D3 preincubated with hsc73 (13D3/HSC73), or  mAb p32 (P32). The cells were chased  with fresh media containing 10% NCS  for 1 h, and then changed to serumsupplemented (solid lines) or serumdeprived (dotted lines) media. Acidsoluble radioactivity in the media was  followed as a measure of proteolysis.  Results shown are the mean for n = 6  (NONE), or n = 4 (13D3, 13D3/ HSC73, and P32). The half-lives (in h)  in the presence and absence of serum,  respectively, are: (A) 80 and 52; (B) 81  and 84; (C) 100 and 74; and (D) 120  and 77.
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Related In: Results  -  Collection

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Figure 6: Effect of mAb 13D3 on enhanced degradation of cellular proteins in response to serum deprivation. Fibroblasts were labeled with [3H]leucine for 2 d, and then incubated overnight in medium containing 10% NCS and either no addition (NONE), or mAb 13D3 (13D3), mAb 13D3 preincubated with hsc73 (13D3/HSC73), or mAb p32 (P32). The cells were chased with fresh media containing 10% NCS for 1 h, and then changed to serumsupplemented (solid lines) or serumdeprived (dotted lines) media. Acidsoluble radioactivity in the media was followed as a measure of proteolysis. Results shown are the mean for n = 6 (NONE), or n = 4 (13D3, 13D3/ HSC73, and P32). The half-lives (in h) in the presence and absence of serum, respectively, are: (A) 80 and 52; (B) 81 and 84; (C) 100 and 74; and (D) 120 and 77.
Mentions: In contrast, the enhanced protein degradation in response to serum withdrawal (Fig. 6 A) was blocked when the mAb was endocytosed alone (Fig. 6 B). Endocytosis of mAb 13D3 previously neutralized with hsc73 (Fig. 6 C), or the control antibody (P32; Fig. 6 D), had little effect. These results were obtained in two additional experiments except that the slightly slower protein degradation observed for the P32-treated cells (Fig. 6 D) was not a consistent finding.

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