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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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Internalized Tfn exhibits prolonged trafficking through punctate structures in hsc70D199S- and hsc70T204V-expressing cells as visualized by epifluorescent microscopy. A 5-min pulse of Alexa488-Tfn (0 min) was chased with unlabeled Tfn and followed for 5, 10, 20, and 30 min. Cells were fixed and processed for fluorescence microscopy as described in Materials and Methods. All images were collected with 2-s exposures. WT, wild type.
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Figure 6: Internalized Tfn exhibits prolonged trafficking through punctate structures in hsc70D199S- and hsc70T204V-expressing cells as visualized by epifluorescent microscopy. A 5-min pulse of Alexa488-Tfn (0 min) was chased with unlabeled Tfn and followed for 5, 10, 20, and 30 min. Cells were fixed and processed for fluorescence microscopy as described in Materials and Methods. All images were collected with 2-s exposures. WT, wild type.

Mentions: Given that the hsc70 ATPase-deficient mutants inhibited CCV uncoating mediated by the wild-type protein in vitro, we expected that overexpression of these mutants might also disrupt CCV disassembly in vivo. We have thus far provided strong evidence that disruption of hsc70 function in vivo alters the steady state distribution of clathrin from the disassembled to assembled states. The potent inhibition of BXX-Tfn recycling would suggest that internalized TfnR might accumulate in newly formed endocytic CCVs. However, when we examined the distribution of TfnR using subcellular fractionation, we were unable to detect an accumulation in the vesicular pool as determined either by differential centrifugation (Fig. 5 A) or by chromatography on S1000 (data not shown). Nor could we detect a TGN-derived cargo molecule, the MPR (data not shown). Therefore, to further elucidate the nature of the recycling defect, we followed the trafficking of a 5-min pulse of Alexa488-Tfn. For these experiments, we studied cells expressing the weaker D199S and T204V hsc70 mutants because they incorporated a significant amount of labeled Tfn within the 5-min pulse time. In control (data not shown) and hsc70WT-expressing cells, internalized Tfn was initially found in punctate structures resembling vesicles but rapidly trafficked to the perinuclear recycling endosome and back out of the cell (Fig. 6). In contrast, the mutant cells exhibited prolonged punctate staining that was still detectable 30 min after internalization. To determine whether these punctate structures were endocytic CCVs, the Alexa488-Tfn–internalized cells were immunolabeled with anti-AP2. Normally, the release of coat proteins from CCVs occurs within 1 min of budding, and punctate structures labeling for both coat protein and internalized Tfn are rare. Accordingly, in control (data not shown) and hsc70WT-expressing cells, AP2 and Alexa488-Tfn colocalize in only a few punctate structures at 5 min after internalization and are completely separated after 20 min (Fig. 7). In contrast, AP2 and Tfn colocalization within punctate structures could be detected even 20 min after internalization in the D199S- and T204V hsc70–expressing cells, suggesting that the uncoating reaction was significantly inhibited (Fig. 7). Similar results were obtained with clathrin labeling and after necessarily longer times of Alexa488-Tfn uptake (30 min) into cells expressing hsc70K71M (data not shown). Surprisingly, in the mutant-expressing cells, only a few costained punctate structures were found even at 5 min after internalization, demonstrating that CCV uncoating is not potently inhibited in these cells. Nonetheless, finding that the majority of Alexa488-Tfn–labeled structures did not costain for AP2 was consistent with the fractionation experiment, demonstrating that assembled AP2–clathrin structures accumulated independently of TfnR. Together these data suggest that the accumulating coated structures might represent empty cages.


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

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

Internalized Tfn exhibits prolonged trafficking through punctate structures in hsc70D199S- and hsc70T204V-expressing cells as visualized by epifluorescent microscopy. A 5-min pulse of Alexa488-Tfn (0 min) was chased with unlabeled Tfn and followed for 5, 10, 20, and 30 min. Cells were fixed and processed for fluorescence microscopy as described in Materials and Methods. All images were collected with 2-s exposures. WT, wild type.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196005&req=5

Figure 6: Internalized Tfn exhibits prolonged trafficking through punctate structures in hsc70D199S- and hsc70T204V-expressing cells as visualized by epifluorescent microscopy. A 5-min pulse of Alexa488-Tfn (0 min) was chased with unlabeled Tfn and followed for 5, 10, 20, and 30 min. Cells were fixed and processed for fluorescence microscopy as described in Materials and Methods. All images were collected with 2-s exposures. WT, wild type.
Mentions: Given that the hsc70 ATPase-deficient mutants inhibited CCV uncoating mediated by the wild-type protein in vitro, we expected that overexpression of these mutants might also disrupt CCV disassembly in vivo. We have thus far provided strong evidence that disruption of hsc70 function in vivo alters the steady state distribution of clathrin from the disassembled to assembled states. The potent inhibition of BXX-Tfn recycling would suggest that internalized TfnR might accumulate in newly formed endocytic CCVs. However, when we examined the distribution of TfnR using subcellular fractionation, we were unable to detect an accumulation in the vesicular pool as determined either by differential centrifugation (Fig. 5 A) or by chromatography on S1000 (data not shown). Nor could we detect a TGN-derived cargo molecule, the MPR (data not shown). Therefore, to further elucidate the nature of the recycling defect, we followed the trafficking of a 5-min pulse of Alexa488-Tfn. For these experiments, we studied cells expressing the weaker D199S and T204V hsc70 mutants because they incorporated a significant amount of labeled Tfn within the 5-min pulse time. In control (data not shown) and hsc70WT-expressing cells, internalized Tfn was initially found in punctate structures resembling vesicles but rapidly trafficked to the perinuclear recycling endosome and back out of the cell (Fig. 6). In contrast, the mutant cells exhibited prolonged punctate staining that was still detectable 30 min after internalization. To determine whether these punctate structures were endocytic CCVs, the Alexa488-Tfn–internalized cells were immunolabeled with anti-AP2. Normally, the release of coat proteins from CCVs occurs within 1 min of budding, and punctate structures labeling for both coat protein and internalized Tfn are rare. Accordingly, in control (data not shown) and hsc70WT-expressing cells, AP2 and Alexa488-Tfn colocalize in only a few punctate structures at 5 min after internalization and are completely separated after 20 min (Fig. 7). In contrast, AP2 and Tfn colocalization within punctate structures could be detected even 20 min after internalization in the D199S- and T204V hsc70–expressing cells, suggesting that the uncoating reaction was significantly inhibited (Fig. 7). Similar results were obtained with clathrin labeling and after necessarily longer times of Alexa488-Tfn uptake (30 min) into cells expressing hsc70K71M (data not shown). Surprisingly, in the mutant-expressing cells, only a few costained punctate structures were found even at 5 min after internalization, demonstrating that CCV uncoating is not potently inhibited in these cells. Nonetheless, finding that the majority of Alexa488-Tfn–labeled structures did not costain for AP2 was consistent with the fractionation experiment, demonstrating that assembled AP2–clathrin structures accumulated independently of TfnR. Together these data suggest that the accumulating coated structures might represent empty cages.

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