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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 is involved in multiple steps in CCV cycling and receptor recycling. Hsc70 releases clathrin from newly budded CCVs (1). Our studies suggest that hsc70 may then be required to chaperone cytosolic clathrin triskelions (2) to allow their functional recruitment to coated pits (3). Within the endosomal compartment, hsc70 also promotes receptor recycling (dashed line, 4).
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Figure 9: Hsc70 is involved in multiple steps in CCV cycling and receptor recycling. Hsc70 releases clathrin from newly budded CCVs (1). Our studies suggest that hsc70 may then be required to chaperone cytosolic clathrin triskelions (2) to allow their functional recruitment to coated pits (3). Within the endosomal compartment, hsc70 also promotes receptor recycling (dashed line, 4).

Mentions: We have characterized several hsc70 ATPase–deficient mutants and found them to dominantly interfere with the CCV cycle in vivo. Within HeLa cells, clathrin is equally distributed between assembled and unassembled pools. Overexpresssion of hsc70 mutants dramatically shifts this equilibrium towards the assembled state, demonstrating that hsc70 plays a role in clathrin dynamics. We observed a significant shift of clathrin and AP1–AP2 complexes into fractions coeluting by size exclusion chromatography with CCVs. We also found prolonged colocalization of AP2 and internalized Tfn in punctate structures, suggesting that the increased assembled pool of clathrin and APs is in part due to a CCV uncoating defect. Hsc70 was previously implicated in releasing clathrin from CCVs based on in vitro studies (Rothman and Schmid 1986). The physiological significance of hsc70-mediated uncoating was supported by microinjection studies (Honing et al. 1994) and by the discovery of a requirement for auxilin, a DnaJ-like protein associated with neuronal CCVs in recruiting hsc70 to assembled clathrin for uncoating (Ungewickell et al. 1995). Recently, nonneuronal (GAK) and yeast (swa2p/aux1p) homologues of auxilin have been identified (Gall et al. 2000; Greener et al. 2000; Pishvaee et al. 2000; Umeda et al. 2000). Deletion of swa2p/aux1p in yeast results in trafficking phenotypes similar to that observed in clathrin-deleted strains, as well as accumulation of coated vesicles (Gall et al. 2000; Pishvaee et al. 2000). The in vivo effects of interference with hsc70 function reported here are complementary to results from yeast and, together, they establish that CCV uncoating is a general cellular function of hsc70 (Fig. 9, step 1).


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

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

Hsc70 is involved in multiple steps in CCV cycling and receptor recycling. Hsc70 releases clathrin from newly budded CCVs (1). Our studies suggest that hsc70 may then be required to chaperone cytosolic clathrin triskelions (2) to allow their functional recruitment to coated pits (3). Within the endosomal compartment, hsc70 also promotes receptor recycling (dashed line, 4).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Hsc70 is involved in multiple steps in CCV cycling and receptor recycling. Hsc70 releases clathrin from newly budded CCVs (1). Our studies suggest that hsc70 may then be required to chaperone cytosolic clathrin triskelions (2) to allow their functional recruitment to coated pits (3). Within the endosomal compartment, hsc70 also promotes receptor recycling (dashed line, 4).
Mentions: We have characterized several hsc70 ATPase–deficient mutants and found them to dominantly interfere with the CCV cycle in vivo. Within HeLa cells, clathrin is equally distributed between assembled and unassembled pools. Overexpresssion of hsc70 mutants dramatically shifts this equilibrium towards the assembled state, demonstrating that hsc70 plays a role in clathrin dynamics. We observed a significant shift of clathrin and AP1–AP2 complexes into fractions coeluting by size exclusion chromatography with CCVs. We also found prolonged colocalization of AP2 and internalized Tfn in punctate structures, suggesting that the increased assembled pool of clathrin and APs is in part due to a CCV uncoating defect. Hsc70 was previously implicated in releasing clathrin from CCVs based on in vitro studies (Rothman and Schmid 1986). The physiological significance of hsc70-mediated uncoating was supported by microinjection studies (Honing et al. 1994) and by the discovery of a requirement for auxilin, a DnaJ-like protein associated with neuronal CCVs in recruiting hsc70 to assembled clathrin for uncoating (Ungewickell et al. 1995). Recently, nonneuronal (GAK) and yeast (swa2p/aux1p) homologues of auxilin have been identified (Gall et al. 2000; Greener et al. 2000; Pishvaee et al. 2000; Umeda et al. 2000). Deletion of swa2p/aux1p in yeast results in trafficking phenotypes similar to that observed in clathrin-deleted strains, as well as accumulation of coated vesicles (Gall et al. 2000; Pishvaee et al. 2000). The in vivo effects of interference with hsc70 function reported here are complementary to results from yeast and, together, they establish that CCV uncoating is a general cellular function of hsc70 (Fig. 9, step 1).

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