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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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Related in: MedlinePlus

Overexpression of hsc70K71M causes endogenous hsc70 to migrate as a complex. Size exclusion chromatography was employed to monitor the potential oligomerization of endogenous hsc70. The independent migration of alcohol dehydrogenase (150 kD) and BSA (66 kD) are shown as markers. Western blotting with 3C5 revealed the migration of endogenous hsc70, hsp70, and the HA-tagged adenovirally expressed mutant and wild-type hsc70 and was quantified through densitometric scanning. WT, wild type.
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Figure 4: Overexpression of hsc70K71M causes endogenous hsc70 to migrate as a complex. Size exclusion chromatography was employed to monitor the potential oligomerization of endogenous hsc70. The independent migration of alcohol dehydrogenase (150 kD) and BSA (66 kD) are shown as markers. Western blotting with 3C5 revealed the migration of endogenous hsc70, hsp70, and the HA-tagged adenovirally expressed mutant and wild-type hsc70 and was quantified through densitometric scanning. WT, wild type.

Mentions: Given that the hsc70 mutants specifically disrupt Tfn uptake and recycling, we were interested in exploring the mechanism by which the endogenous protein was inhibited. We hypothesized that these mutants could inhibit through (a) sequestration of clathrin, (b) sequestration of auxilin, the DnaJ protein that is thought to target hsc70 to clathrin, and/or (c) sequestration of endogenous hsc70. In vitro, hsc70 forms a stable complex with clathrin and auxilin (Schmid and Rothman 1985; Holstein et al. 1996); however, we were unable to detect similar complexes with the hsc70 mutants (Newmyer, S.L., and S.L. Schmid, manuscript in preparation), suggesting that inhibition is not due to sequestration of either clathrin or auxilin. In contrast, the mutant hsc70s could form dimers and higher order oligomers with hsc70WT as detected through crosslinking (data not shown). Hsc70 oligomerization is promoted in the presence of ADP, and it has been suggested that oligomerization inhibits hsc70 activity (Benaroudj et al. 1996; Gao et al. 1996). Given that our mutants have aberrant ATPase cycles, we looked for their ability to oligomerize with endogenous hsc70 by using size exclusion chromatography to monitor the presence of such complexes in the hsc70 expressing cells (Fig. 4). In the hsc70WT-expressing cells (solid line), the endogenous and HA-tagged proteins coeluted just before the BSA standard. Incubation of the lysate with ATP did not alter this elution profile (data not shown), suggesting that under these conditions hsc70 existed as a monomer (Angelidis et al. 1999). Strikingly, in cells overexpressing the K71M mutant, endogenous hsc70 coeluted with hsc70K71M as a higher molecular weight species (dashed line), suggesting that these proteins exist in a complex. We could not determine whether other cytosolic proteins were also present in this complex. As with hsc70WT cells, the elution profiles observed were unaffected by incubation under ATP-depleting and -supplementing conditions, indicating that the faster migrating hsc70 species is stable to the addition of ATP (data not shown). These results suggested that the overexpression of hsc70 ATPaseā€“deficient mutants created a new equilibrium between active monomeric and potentially inactive oligomeric hsc70 species and that by overexpressing these mutants we have, in effect, sequestered the endogenous protein.


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

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

Overexpression of hsc70K71M causes endogenous hsc70 to migrate as a complex. Size exclusion chromatography was employed to monitor the potential oligomerization of endogenous hsc70. The independent migration of alcohol dehydrogenase (150 kD) and BSA (66 kD) are shown as markers. Western blotting with 3C5 revealed the migration of endogenous hsc70, hsp70, and the HA-tagged adenovirally expressed mutant and wild-type hsc70 and was quantified through densitometric scanning. WT, wild type.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Overexpression of hsc70K71M causes endogenous hsc70 to migrate as a complex. Size exclusion chromatography was employed to monitor the potential oligomerization of endogenous hsc70. The independent migration of alcohol dehydrogenase (150 kD) and BSA (66 kD) are shown as markers. Western blotting with 3C5 revealed the migration of endogenous hsc70, hsp70, and the HA-tagged adenovirally expressed mutant and wild-type hsc70 and was quantified through densitometric scanning. WT, wild type.
Mentions: Given that the hsc70 mutants specifically disrupt Tfn uptake and recycling, we were interested in exploring the mechanism by which the endogenous protein was inhibited. We hypothesized that these mutants could inhibit through (a) sequestration of clathrin, (b) sequestration of auxilin, the DnaJ protein that is thought to target hsc70 to clathrin, and/or (c) sequestration of endogenous hsc70. In vitro, hsc70 forms a stable complex with clathrin and auxilin (Schmid and Rothman 1985; Holstein et al. 1996); however, we were unable to detect similar complexes with the hsc70 mutants (Newmyer, S.L., and S.L. Schmid, manuscript in preparation), suggesting that inhibition is not due to sequestration of either clathrin or auxilin. In contrast, the mutant hsc70s could form dimers and higher order oligomers with hsc70WT as detected through crosslinking (data not shown). Hsc70 oligomerization is promoted in the presence of ADP, and it has been suggested that oligomerization inhibits hsc70 activity (Benaroudj et al. 1996; Gao et al. 1996). Given that our mutants have aberrant ATPase cycles, we looked for their ability to oligomerize with endogenous hsc70 by using size exclusion chromatography to monitor the presence of such complexes in the hsc70 expressing cells (Fig. 4). In the hsc70WT-expressing cells (solid line), the endogenous and HA-tagged proteins coeluted just before the BSA standard. Incubation of the lysate with ATP did not alter this elution profile (data not shown), suggesting that under these conditions hsc70 existed as a monomer (Angelidis et al. 1999). Strikingly, in cells overexpressing the K71M mutant, endogenous hsc70 coeluted with hsc70K71M as a higher molecular weight species (dashed line), suggesting that these proteins exist in a complex. We could not determine whether other cytosolic proteins were also present in this complex. As with hsc70WT cells, the elution profiles observed were unaffected by incubation under ATP-depleting and -supplementing conditions, indicating that the faster migrating hsc70 species is stable to the addition of ATP (data not shown). These results suggested that the overexpression of hsc70 ATPaseā€“deficient mutants created a new equilibrium between active monomeric and potentially inactive oligomeric hsc70 species and that by overexpressing these mutants we have, in effect, sequestered the endogenous protein.

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