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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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Distribution of ly-hsc73 and lgp120 by confocal microscopy using a narrow optical section. Fibroblasts were serum deprived overnight, methanol fixed, and then incubated with mAb  13D3 and anti-lgp120 simultaneously followed by Texas red– and  fluorescein-conjugated second antibodies to reveal ly-hsc73 (green)  and lgp120 (red). The thickness of the optical section analyzed  was 0.09 μm. Colocalization of both proteins registers as yellow/ orange. (Insets) Left panel, fluorescein channel; right panel,  Texas red channel. Bar, 10 μm.
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Figure 2: Distribution of ly-hsc73 and lgp120 by confocal microscopy using a narrow optical section. Fibroblasts were serum deprived overnight, methanol fixed, and then incubated with mAb 13D3 and anti-lgp120 simultaneously followed by Texas red– and fluorescein-conjugated second antibodies to reveal ly-hsc73 (green) and lgp120 (red). The thickness of the optical section analyzed was 0.09 μm. Colocalization of both proteins registers as yellow/ orange. (Insets) Left panel, fluorescein channel; right panel, Texas red channel. Bar, 10 μm.

Mentions: We examined and quantitated the lysosomal localization of ly-hsc73 further by confocal microscopy. Double fluorescence staining of fibroblasts was carried out with mAb 13D3 and anti-lgp120 antibodies (Figs. 2 and 3). lgp120 is the major glycoprotein in lysosomal membranes (Green et al., 1987). As previously described, hsc73 is an abundant cytosolic protein (Giebel et al., 1988). To avoid the interference of cytosolic hsc73 during immunolabeling with mAb 13D3, cells were fixed in cold methanol, which did not fix the cytosolic fraction of hsc73 and allowed us to examine organelle-associated hsp70s that are recognized by mAb 13D3. The mAb 13D3 (green signal) largely colocalized with the anti-lgp120 antibodies (red signal). A thin optical section (0.09 μm) shows that both proteins are confined to the cytoplasmic compartment (Fig. 2). Colocalization in the merged images (Figs. 2 and 3) registers as orange/yellow.


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

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

Distribution of ly-hsc73 and lgp120 by confocal microscopy using a narrow optical section. Fibroblasts were serum deprived overnight, methanol fixed, and then incubated with mAb  13D3 and anti-lgp120 simultaneously followed by Texas red– and  fluorescein-conjugated second antibodies to reveal ly-hsc73 (green)  and lgp120 (red). The thickness of the optical section analyzed  was 0.09 μm. Colocalization of both proteins registers as yellow/ orange. (Insets) Left panel, fluorescein channel; right panel,  Texas red channel. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Distribution of ly-hsc73 and lgp120 by confocal microscopy using a narrow optical section. Fibroblasts were serum deprived overnight, methanol fixed, and then incubated with mAb 13D3 and anti-lgp120 simultaneously followed by Texas red– and fluorescein-conjugated second antibodies to reveal ly-hsc73 (green) and lgp120 (red). The thickness of the optical section analyzed was 0.09 μm. Colocalization of both proteins registers as yellow/ orange. (Insets) Left panel, fluorescein channel; right panel, Texas red channel. Bar, 10 μm.
Mentions: We examined and quantitated the lysosomal localization of ly-hsc73 further by confocal microscopy. Double fluorescence staining of fibroblasts was carried out with mAb 13D3 and anti-lgp120 antibodies (Figs. 2 and 3). lgp120 is the major glycoprotein in lysosomal membranes (Green et al., 1987). As previously described, hsc73 is an abundant cytosolic protein (Giebel et al., 1988). To avoid the interference of cytosolic hsc73 during immunolabeling with mAb 13D3, cells were fixed in cold methanol, which did not fix the cytosolic fraction of hsc73 and allowed us to examine organelle-associated hsp70s that are recognized by mAb 13D3. The mAb 13D3 (green signal) largely colocalized with the anti-lgp120 antibodies (red signal). A thin optical section (0.09 μm) shows that both proteins are confined to the cytoplasmic compartment (Fig. 2). Colocalization in the merged images (Figs. 2 and 3) registers as orange/yellow.

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