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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 in serum-deprived and serumsupplemented fibroblasts using a thick  optical section. Methanol-fixed fibroblasts were stained with primary antibodies to hsc73 (green) and lgp120  (red) as described in the legend to Fig.  2. The merged color images using an  optical section of 4 μm show: (A) serum-deprived fibroblast and (B) serum-supplemented fibroblast. Note  that lysosomes appear to fuse, forming  a tubular network when cells are serum  deprived (A). Bar, 10 μm.
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Figure 3: Distribution of ly-hsc73 and lgp120 in serum-deprived and serumsupplemented fibroblasts using a thick optical section. Methanol-fixed fibroblasts were stained with primary antibodies to hsc73 (green) and lgp120 (red) as described in the legend to Fig. 2. The merged color images using an optical section of 4 μm show: (A) serum-deprived fibroblast and (B) serum-supplemented fibroblast. Note that lysosomes appear to fuse, forming a tubular network when cells are serum deprived (A). Bar, 10 μm.

Mentions: The cells grown on glass coverslips were washed five times with serumfree culture medium, and then fixed in −20°C methanol for 1 min and stored at 4°C in PBS containing 0.02% NaN3. For fluorescent double labeling of ly-hsc73 and lgp120, all incubations and washes were at 25°C. Primary antibodies were diluted 1:50, and secondary antibodies were diluted 1:100 in PBS containing 0.1% BSA and 0.02% NaN3. Cells were incubated for 1 h with a mixture of primary antibodies, washed 10 times in PBS, and incubated for 1 h with each of the corresponding secondary antibodies. Coverslips were mounted in mounting media containing an antibleaching agent (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Images were collected on a laser scanning confocal microscope, either an MRC 600 (Bio Rad Laboratories) or an ODYSSEY XL (NORAN Instruments, Middleton, WI). The image analysis software used was INTERVISION (NORAN Instruments). Pictures were taken with Kodachrome 64 and TMAX400 film (Eastman Kodak Co.). Fig. 3 C was generated using Adobe Photoshop 3.0 software (Adobe Systems Inc., Mountain View, CA). No fluorescence was associated with cells after incubation with secondary antibodies alone (data not shown).


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 in serum-deprived and serumsupplemented fibroblasts using a thick  optical section. Methanol-fixed fibroblasts were stained with primary antibodies to hsc73 (green) and lgp120  (red) as described in the legend to Fig.  2. The merged color images using an  optical section of 4 μm show: (A) serum-deprived fibroblast and (B) serum-supplemented fibroblast. Note  that lysosomes appear to fuse, forming  a tubular network when cells are serum  deprived (A). Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139836&req=5

Figure 3: Distribution of ly-hsc73 and lgp120 in serum-deprived and serumsupplemented fibroblasts using a thick optical section. Methanol-fixed fibroblasts were stained with primary antibodies to hsc73 (green) and lgp120 (red) as described in the legend to Fig. 2. The merged color images using an optical section of 4 μm show: (A) serum-deprived fibroblast and (B) serum-supplemented fibroblast. Note that lysosomes appear to fuse, forming a tubular network when cells are serum deprived (A). Bar, 10 μm.
Mentions: The cells grown on glass coverslips were washed five times with serumfree culture medium, and then fixed in −20°C methanol for 1 min and stored at 4°C in PBS containing 0.02% NaN3. For fluorescent double labeling of ly-hsc73 and lgp120, all incubations and washes were at 25°C. Primary antibodies were diluted 1:50, and secondary antibodies were diluted 1:100 in PBS containing 0.1% BSA and 0.02% NaN3. Cells were incubated for 1 h with a mixture of primary antibodies, washed 10 times in PBS, and incubated for 1 h with each of the corresponding secondary antibodies. Coverslips were mounted in mounting media containing an antibleaching agent (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Images were collected on a laser scanning confocal microscope, either an MRC 600 (Bio Rad Laboratories) or an ODYSSEY XL (NORAN Instruments, Middleton, WI). The image analysis software used was INTERVISION (NORAN Instruments). Pictures were taken with Kodachrome 64 and TMAX400 film (Eastman Kodak Co.). Fig. 3 C was generated using Adobe Photoshop 3.0 software (Adobe Systems Inc., Mountain View, CA). No fluorescence was associated with cells after incubation with secondary antibodies alone (data not shown).

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