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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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Model representing the roles of hsc73 and ly-hsc73 in  the selective lysosomal protein degradation pathway. The protein  substrate depicted is RNase A, and the black rectangle represents  the KFERQ sequence. Steps 1–4 are as described in the text.
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Figure 9: Model representing the roles of hsc73 and ly-hsc73 in the selective lysosomal protein degradation pathway. The protein substrate depicted is RNase A, and the black rectangle represents the KFERQ sequence. Steps 1–4 are as described in the text.

Mentions: These results together with previously defined features of the hsc73-stimulated pathway of lysosomal proteolysis lead us to the working model shown in Fig. 9. Cytosolic hsc73, acting as a molecular chaperone, recognizes specific sequences (KFERQ and related peptides) in proteins that are targeted for lysosomal degradation in response to serum withdrawal. These sequences may be exposed by changes in the conformation of the substrate proteins or by alterations in protein–protein interactions. Alternatively, cytosolic hsc73 may be altered by serum withdrawal in some way so that it now acts in the lysosomal degradative pathway rather than in organelle synthetic pathways or in the uncoating of clathrin-coated vesicles.


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

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

Model representing the roles of hsc73 and ly-hsc73 in  the selective lysosomal protein degradation pathway. The protein  substrate depicted is RNase A, and the black rectangle represents  the KFERQ sequence. Steps 1–4 are as described in the text.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139836&req=5

Figure 9: Model representing the roles of hsc73 and ly-hsc73 in the selective lysosomal protein degradation pathway. The protein substrate depicted is RNase A, and the black rectangle represents the KFERQ sequence. Steps 1–4 are as described in the text.
Mentions: These results together with previously defined features of the hsc73-stimulated pathway of lysosomal proteolysis lead us to the working model shown in Fig. 9. Cytosolic hsc73, acting as a molecular chaperone, recognizes specific sequences (KFERQ and related peptides) in proteins that are targeted for lysosomal degradation in response to serum withdrawal. These sequences may be exposed by changes in the conformation of the substrate proteins or by alterations in protein–protein interactions. Alternatively, cytosolic hsc73 may be altered by serum withdrawal in some way so that it now acts in the lysosomal degradative pathway rather than in organelle synthetic pathways or in the uncoating of clathrin-coated vesicles.

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