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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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Two-dimensional gel electrophoresis of hsc73 and lyhsc73. Fibroblast cytosol (150 μg protein; A), a mixture of fibroblast lysosomes (200 μg protein) and purified bovine brain hsc73  (1 μg protein; B), fibroblast lysosomes (200 μg protein), and fibroblast lysosomal membranes (100 μg protein; C) were separated as described in Materials and Methods. The membranes  were immunoblotted with mAb 13D3 and developed with the  ECL system. (Arrows) hsc73.
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Figure 5: Two-dimensional gel electrophoresis of hsc73 and lyhsc73. Fibroblast cytosol (150 μg protein; A), a mixture of fibroblast lysosomes (200 μg protein) and purified bovine brain hsc73 (1 μg protein; B), fibroblast lysosomes (200 μg protein), and fibroblast lysosomal membranes (100 μg protein; C) were separated as described in Materials and Methods. The membranes were immunoblotted with mAb 13D3 and developed with the ECL system. (Arrows) hsc73.

Mentions: This luminal localization of the majority of ly-hsc73 is further supported by our two-dimensional electrophoresis analyses followed by immunoblotting with mAb 13D3. Fig. 5 A shows the multiple isoforms of fibroblast cytosolic hsc73. Fig. 5 B represents mixed ly-hsc73 and bovine brain hsc73 purified as described previously (Welsh and Feramisco, 1985). Fibroblast cytosol and bovine brain cytosol showed similar isoforms of hsc73 (data not shown). Fig. 5 C shows that the isoelectric point of the ly-hsc73 corresponds to the most acidic isoform present in the cytosol. A longer exposure of the immunoblot allowed us to visualize the presence of a small amount of the most abundant cytoplasmic hsc73 isoform (data not shown). This hsc73 isoform was localized to the lysosomal membrane (Fig. 5 D). The cause of these charge variants is not known, but such isoforms have also been reported for other cell types (Watowich and Morimoto, 1988; Bhattacharyya et al., 1995).


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

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

Two-dimensional gel electrophoresis of hsc73 and lyhsc73. Fibroblast cytosol (150 μg protein; A), a mixture of fibroblast lysosomes (200 μg protein) and purified bovine brain hsc73  (1 μg protein; B), fibroblast lysosomes (200 μg protein), and fibroblast lysosomal membranes (100 μg protein; C) were separated as described in Materials and Methods. The membranes  were immunoblotted with mAb 13D3 and developed with the  ECL system. (Arrows) hsc73.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Two-dimensional gel electrophoresis of hsc73 and lyhsc73. Fibroblast cytosol (150 μg protein; A), a mixture of fibroblast lysosomes (200 μg protein) and purified bovine brain hsc73 (1 μg protein; B), fibroblast lysosomes (200 μg protein), and fibroblast lysosomal membranes (100 μg protein; C) were separated as described in Materials and Methods. The membranes were immunoblotted with mAb 13D3 and developed with the ECL system. (Arrows) hsc73.
Mentions: This luminal localization of the majority of ly-hsc73 is further supported by our two-dimensional electrophoresis analyses followed by immunoblotting with mAb 13D3. Fig. 5 A shows the multiple isoforms of fibroblast cytosolic hsc73. Fig. 5 B represents mixed ly-hsc73 and bovine brain hsc73 purified as described previously (Welsh and Feramisco, 1985). Fibroblast cytosol and bovine brain cytosol showed similar isoforms of hsc73 (data not shown). Fig. 5 C shows that the isoelectric point of the ly-hsc73 corresponds to the most acidic isoform present in the cytosol. A longer exposure of the immunoblot allowed us to visualize the presence of a small amount of the most abundant cytoplasmic hsc73 isoform (data not shown). This hsc73 isoform was localized to the lysosomal membrane (Fig. 5 D). The cause of these charge variants is not known, but such isoforms have also been reported for other cell types (Watowich and Morimoto, 1988; Bhattacharyya et al., 1995).

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