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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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Hsc70 ATPase domain mutants inhibit hsc70-mediated clathrin uncoating. CCVs were incubated in the presence of wild-type and mutant hsc70 proteins. (A) Shown is a Coomassie blue–stained gel of clathrin released into the 100,000-g supernatant. The final μM concentration of recombinant protein used is indicated in parenthesis. (B) The uncoating reaction mediated by hsc70Native (0.4 μM) was assayed in the presence of increasing hsc70 mutant concentration. The error bars reflect the SD from three independent experiments. WT, wild type.
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Figure 1: Hsc70 ATPase domain mutants inhibit hsc70-mediated clathrin uncoating. CCVs were incubated in the presence of wild-type and mutant hsc70 proteins. (A) Shown is a Coomassie blue–stained gel of clathrin released into the 100,000-g supernatant. The final μM concentration of recombinant protein used is indicated in parenthesis. (B) The uncoating reaction mediated by hsc70Native (0.4 μM) was assayed in the presence of increasing hsc70 mutant concentration. The error bars reflect the SD from three independent experiments. WT, wild type.

Mentions: None of the hsc70 ATPase domain mutants could efficiently release clathrin from CCVs, even when present at fivefold higher concentrations compared with the recombinant wild-type protein (Fig. 1 A). Thus, as has been observed in vitro with hsp70 ATPase domain mutants (Rajapandi et al. 1998), proper ATPase cycling was required for hsc70-mediated CCV uncoating activity. To learn whether the hsc70 mutants could be used in vivo to disrupt hsc70 function, the mutant proteins were titrated into the CCV uncoating reaction mediated by native hsc70, purified from brain cytosol. All three of the mutants interfered with the ability of hsc70Native to mediate clathrin release (Fig. 1 B). Inhibition of the uncoating reaction occurred in a saturable manner with half maximal inhibition occurring at approximately fourfold excess of either hsc70K71M or hsc70T204V to hsc70Native. Hsc70D199S also inhibited hsc70Native-mediated clathrin release, though at higher concentrations. Thus, these mutants have dominant-interfering effects and can be used to probe hsc70 function in the cell.


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

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

Hsc70 ATPase domain mutants inhibit hsc70-mediated clathrin uncoating. CCVs were incubated in the presence of wild-type and mutant hsc70 proteins. (A) Shown is a Coomassie blue–stained gel of clathrin released into the 100,000-g supernatant. The final μM concentration of recombinant protein used is indicated in parenthesis. (B) The uncoating reaction mediated by hsc70Native (0.4 μM) was assayed in the presence of increasing hsc70 mutant concentration. The error bars reflect the SD from three independent experiments. WT, wild type.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Hsc70 ATPase domain mutants inhibit hsc70-mediated clathrin uncoating. CCVs were incubated in the presence of wild-type and mutant hsc70 proteins. (A) Shown is a Coomassie blue–stained gel of clathrin released into the 100,000-g supernatant. The final μM concentration of recombinant protein used is indicated in parenthesis. (B) The uncoating reaction mediated by hsc70Native (0.4 μM) was assayed in the presence of increasing hsc70 mutant concentration. The error bars reflect the SD from three independent experiments. WT, wild type.
Mentions: None of the hsc70 ATPase domain mutants could efficiently release clathrin from CCVs, even when present at fivefold higher concentrations compared with the recombinant wild-type protein (Fig. 1 A). Thus, as has been observed in vitro with hsp70 ATPase domain mutants (Rajapandi et al. 1998), proper ATPase cycling was required for hsc70-mediated CCV uncoating activity. To learn whether the hsc70 mutants could be used in vivo to disrupt hsc70 function, the mutant proteins were titrated into the CCV uncoating reaction mediated by native hsc70, purified from brain cytosol. All three of the mutants interfered with the ability of hsc70Native to mediate clathrin release (Fig. 1 B). Inhibition of the uncoating reaction occurred in a saturable manner with half maximal inhibition occurring at approximately fourfold excess of either hsc70K71M or hsc70T204V to hsc70Native. Hsc70D199S also inhibited hsc70Native-mediated clathrin release, though at higher concentrations. Thus, these mutants have dominant-interfering effects and can be used to probe hsc70 function in the cell.

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