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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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Subcellular fractionation of hsc70WT- and hsc70K71M-expressing and control cells. (A) Western blot analysis of the crude subcellular distribution of coat proteins and receptors includes the total cellular (T), the 14,000-g pelleted heavy membrane (P1), the 100,000-g pelleted vesicular membrane (P2), and the cytosolic (S) pools. (B) Western blot analysis of fractions 17–31 collected from an S-1000 column show the migration of vesicular and cytosolic proteins within the low speed supernatant. The migration of purified CCVs and clathrin is indicated. Anticlathrin TD1, anti-β1/β2 adaptin 100/1, anti–δ adaptin, and anti–TfnR D65 were used for immunoblotting. WT, wild type.
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Figure 5: Subcellular fractionation of hsc70WT- and hsc70K71M-expressing and control cells. (A) Western blot analysis of the crude subcellular distribution of coat proteins and receptors includes the total cellular (T), the 14,000-g pelleted heavy membrane (P1), the 100,000-g pelleted vesicular membrane (P2), and the cytosolic (S) pools. (B) Western blot analysis of fractions 17–31 collected from an S-1000 column show the migration of vesicular and cytosolic proteins within the low speed supernatant. The migration of purified CCVs and clathrin is indicated. Anticlathrin TD1, anti-β1/β2 adaptin 100/1, anti–δ adaptin, and anti–TfnR D65 were used for immunoblotting. WT, wild type.

Mentions: We next tested whether CCVs indeed accumulate in hsc70 mutant expressing cells as suggested by our observed recycling defect. To examine this, we performed subcellular fractionation of control and hsc70-expressing cells. Lysed cells were fractionated by low and high speed centrifugation to obtain pellets containing heavy membrane (P1)– and vesicular membrane (P2)–associated proteins, respectively, and the remaining supernatant was used to identify cytosolic protein (S). In control and hsc70WT-expressing cells, clathrin was distributed evenly between the heavy membrane and cytosolic pools (Fig. 5 A). Strikingly, in hsc70K71M-expressing cells, the majority of the cytosolic pool of coat proteins was lost, and the vesicular pool correspondingly increased. Similar results were observed in hsc70D199S- and hsc70T204V-expressing cells, with the degree of accumulation of clathrin in the P2 pool being proportional to the relative inhibitory strengths of the mutants (i.e., K71M > T204V > D199S; data not shown). To further characterize the vesicular versus cytosolic pools, the low speed supernatant was fractionated by size exclusion chromatography to resolve triskelions from assembled clathrin cages. The majority of clathrin migrated as triskelions in control and hsc70WT-expressing cells but, consistent with our subcellular fractionation findings, migrated like assembled clathrin in the hsc70K71M-expressing cells (Fig. 5 B). A strong uncoating defect would predict such an increased CCV phenotype. Moreover, AP1 and AP2 cofractionated with clathrin in the hsc70K71M-expressing cells as would be expected if uncoating were inhibited. Recall that hsc70 is required for adaptor protein (AP) release from purified CCVs (Hannan et al. 1998). We also looked at AP3, which has been implicated in clathrin-dependent and -independent sorting at the TGN and endosomes (Simpson et al. 1996; Dell'Angelica et al. 1998). In contrast to AP1 and AP2, we find that AP3 fractionated independently of the assembled pool of clathrin in hsc70K71M-expressing cells (Fig. 5A and Fig. B). Relative to control and hsc70WT-expressing cells, AP3 was not increased in the high speed pellet, and its migration on the S-1000 column was not altered, consistent with reports that AP3 can function independently of clathrin (Faundez and Kelly 2000). Disruption of hsc70 activity, therefore, causes a striking redistribution of clathrin, AP1, and AP2 to an assembled pool that is consistent with there being a block in uncoating of the corresponding TGN-, endosome-, and PM-derived CCVs.


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

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

Subcellular fractionation of hsc70WT- and hsc70K71M-expressing and control cells. (A) Western blot analysis of the crude subcellular distribution of coat proteins and receptors includes the total cellular (T), the 14,000-g pelleted heavy membrane (P1), the 100,000-g pelleted vesicular membrane (P2), and the cytosolic (S) pools. (B) Western blot analysis of fractions 17–31 collected from an S-1000 column show the migration of vesicular and cytosolic proteins within the low speed supernatant. The migration of purified CCVs and clathrin is indicated. Anticlathrin TD1, anti-β1/β2 adaptin 100/1, anti–δ adaptin, and anti–TfnR D65 were used for immunoblotting. WT, wild type.
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Figure 5: Subcellular fractionation of hsc70WT- and hsc70K71M-expressing and control cells. (A) Western blot analysis of the crude subcellular distribution of coat proteins and receptors includes the total cellular (T), the 14,000-g pelleted heavy membrane (P1), the 100,000-g pelleted vesicular membrane (P2), and the cytosolic (S) pools. (B) Western blot analysis of fractions 17–31 collected from an S-1000 column show the migration of vesicular and cytosolic proteins within the low speed supernatant. The migration of purified CCVs and clathrin is indicated. Anticlathrin TD1, anti-β1/β2 adaptin 100/1, anti–δ adaptin, and anti–TfnR D65 were used for immunoblotting. WT, wild type.
Mentions: We next tested whether CCVs indeed accumulate in hsc70 mutant expressing cells as suggested by our observed recycling defect. To examine this, we performed subcellular fractionation of control and hsc70-expressing cells. Lysed cells were fractionated by low and high speed centrifugation to obtain pellets containing heavy membrane (P1)– and vesicular membrane (P2)–associated proteins, respectively, and the remaining supernatant was used to identify cytosolic protein (S). In control and hsc70WT-expressing cells, clathrin was distributed evenly between the heavy membrane and cytosolic pools (Fig. 5 A). Strikingly, in hsc70K71M-expressing cells, the majority of the cytosolic pool of coat proteins was lost, and the vesicular pool correspondingly increased. Similar results were observed in hsc70D199S- and hsc70T204V-expressing cells, with the degree of accumulation of clathrin in the P2 pool being proportional to the relative inhibitory strengths of the mutants (i.e., K71M > T204V > D199S; data not shown). To further characterize the vesicular versus cytosolic pools, the low speed supernatant was fractionated by size exclusion chromatography to resolve triskelions from assembled clathrin cages. The majority of clathrin migrated as triskelions in control and hsc70WT-expressing cells but, consistent with our subcellular fractionation findings, migrated like assembled clathrin in the hsc70K71M-expressing cells (Fig. 5 B). A strong uncoating defect would predict such an increased CCV phenotype. Moreover, AP1 and AP2 cofractionated with clathrin in the hsc70K71M-expressing cells as would be expected if uncoating were inhibited. Recall that hsc70 is required for adaptor protein (AP) release from purified CCVs (Hannan et al. 1998). We also looked at AP3, which has been implicated in clathrin-dependent and -independent sorting at the TGN and endosomes (Simpson et al. 1996; Dell'Angelica et al. 1998). In contrast to AP1 and AP2, we find that AP3 fractionated independently of the assembled pool of clathrin in hsc70K71M-expressing cells (Fig. 5A and Fig. B). Relative to control and hsc70WT-expressing cells, AP3 was not increased in the high speed pellet, and its migration on the S-1000 column was not altered, consistent with reports that AP3 can function independently of clathrin (Faundez and Kelly 2000). Disruption of hsc70 activity, therefore, causes a striking redistribution of clathrin, AP1, and AP2 to an assembled pool that is consistent with there being a block in uncoating of the corresponding TGN-, endosome-, and PM-derived CCVs.

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