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Dominant-interfering Hsc70 mutants disrupt multiple stages of the clathrin-coated vesicle cycle in vivo.

Newmyer SL, Schmid SL - J. Cell Biol. (2001)

Bottom Line: The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs.These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments.Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.

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Steady state distribution of TfnR and MPR in wild-type and mutant hsc70–expressing cells. The cellular localization of PM- and TGN-cycled receptors were visualized by epifluorescent microscopy. Anti–TfnR D65 and anti-MPR antibodies were used to label adenovirally infected cells after their fixation. Arrows indicate the tubular TfnR-labeled structures observed in hsc70K71M-expressing cells. WT, wild type.
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Figure 8: Steady state distribution of TfnR and MPR in wild-type and mutant hsc70–expressing cells. The cellular localization of PM- and TGN-cycled receptors were visualized by epifluorescent microscopy. Anti–TfnR D65 and anti-MPR antibodies were used to label adenovirally infected cells after their fixation. Arrows indicate the tubular TfnR-labeled structures observed in hsc70K71M-expressing cells. WT, wild type.

Mentions: The overexpression of hsc70 ATPase mutants significantly inhibited the trafficking of Tfn, suggesting that the trafficking of the receptor was also inhibited. Therefore, we monitored the steady state distribution of TfnR by immunofluorescence to learn how its trafficking was affected by the altered endocytic and recycling kinetics observed for the hsc70 mutant–expressing cells. Accumulation of TfnR within the recycling compartment was decreased, corresponding to the increased inhibitory potency of the hsc70 mutant (Fig. 8). This was especially evident in cells expressing hsc70K71M where TfnR appeared to accumulate in vesicular and tubular structures (arrows).


Dominant-interfering Hsc70 mutants disrupt multiple stages of the clathrin-coated vesicle cycle in vivo.

Newmyer SL, Schmid SL - J. Cell Biol. (2001)

Steady state distribution of TfnR and MPR in wild-type and mutant hsc70–expressing cells. The cellular localization of PM- and TGN-cycled receptors were visualized by epifluorescent microscopy. Anti–TfnR D65 and anti-MPR antibodies were used to label adenovirally infected cells after their fixation. Arrows indicate the tubular TfnR-labeled structures observed in hsc70K71M-expressing cells. WT, wild type.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196005&req=5

Figure 8: Steady state distribution of TfnR and MPR in wild-type and mutant hsc70–expressing cells. The cellular localization of PM- and TGN-cycled receptors were visualized by epifluorescent microscopy. Anti–TfnR D65 and anti-MPR antibodies were used to label adenovirally infected cells after their fixation. Arrows indicate the tubular TfnR-labeled structures observed in hsc70K71M-expressing cells. WT, wild type.
Mentions: The overexpression of hsc70 ATPase mutants significantly inhibited the trafficking of Tfn, suggesting that the trafficking of the receptor was also inhibited. Therefore, we monitored the steady state distribution of TfnR by immunofluorescence to learn how its trafficking was affected by the altered endocytic and recycling kinetics observed for the hsc70 mutant–expressing cells. Accumulation of TfnR within the recycling compartment was decreased, corresponding to the increased inhibitory potency of the hsc70 mutant (Fig. 8). This was especially evident in cells expressing hsc70K71M where TfnR appeared to accumulate in vesicular and tubular structures (arrows).

Bottom Line: The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs.These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments.Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.

Show MeSH
Related in: MedlinePlus