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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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The trafficking of newly synthesized TfnR is slightly inhibited by dominant-interfering hsc70 mutants. Autoradiogram (A) and corresponding quantitation (B and C) of immunoprecipitated TfnR from a representative experiment. (B) Trafficking of TfnR from the ER (eHs) to the cis-Golgi region (eHr) was monitored through the resistance to Endo H. (C) Trafficking to the trans-Golgi region was assayed through the shift of glycosylation from the immature (i) to mature (m) receptor state. WT, wild type.
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Figure 3: The trafficking of newly synthesized TfnR is slightly inhibited by dominant-interfering hsc70 mutants. Autoradiogram (A) and corresponding quantitation (B and C) of immunoprecipitated TfnR from a representative experiment. (B) Trafficking of TfnR from the ER (eHs) to the cis-Golgi region (eHr) was monitored through the resistance to Endo H. (C) Trafficking to the trans-Golgi region was assayed through the shift of glycosylation from the immature (i) to mature (m) receptor state. WT, wild type.

Mentions: Given the many possible functions of hsc70 within the cell, overexpression of dominant-interfering hsc70 mutants might have a global effect on cell viability. Therefore, to learn whether the inhibitory effects of hsc70 ATPase domain mutants on endocytosis and recycling were specific, we determined the effects of overexpression of hsc70WT or hsc70K71M, the most potent mutant, on vesicular trafficking along the secretory pathway. Cells were pulse labeled with [35S]methionine, and the delivery of newly synthesized TfnR from the ER to cis- and trans-Golgi compartments was monitored by acquisition of Endo H resistance and the addition of complex oligosaccharides, respectively. Quantitation of the results shown in Fig. 3 A reveal that Endo H–resistant (Fig. 3 B) and mature (Fig. 3 C) forms of TfnR appeared at similar rates in control and hsc70WT-expressing cells. The corresponding rates observed in hsc70K71M-expressing cells were slightly inhibited (approximately twofold). The rates of biosynthesis and translocation of TfnR into the ER, as assessed by radiolabel incorporation and addition of core oligosaccharides, were also not significantly inhibited with overexpression of hsc70K71M (data not shown). Thus, TfnR biosynthesis was only slightly inhibited by the most potent inhibitor of BXX-Tfn uptake and recycling.


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

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

The trafficking of newly synthesized TfnR is slightly inhibited by dominant-interfering hsc70 mutants. Autoradiogram (A) and corresponding quantitation (B and C) of immunoprecipitated TfnR from a representative experiment. (B) Trafficking of TfnR from the ER (eHs) to the cis-Golgi region (eHr) was monitored through the resistance to Endo H. (C) Trafficking to the trans-Golgi region was assayed through the shift of glycosylation from the immature (i) to mature (m) receptor state. WT, wild type.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: The trafficking of newly synthesized TfnR is slightly inhibited by dominant-interfering hsc70 mutants. Autoradiogram (A) and corresponding quantitation (B and C) of immunoprecipitated TfnR from a representative experiment. (B) Trafficking of TfnR from the ER (eHs) to the cis-Golgi region (eHr) was monitored through the resistance to Endo H. (C) Trafficking to the trans-Golgi region was assayed through the shift of glycosylation from the immature (i) to mature (m) receptor state. WT, wild type.
Mentions: Given the many possible functions of hsc70 within the cell, overexpression of dominant-interfering hsc70 mutants might have a global effect on cell viability. Therefore, to learn whether the inhibitory effects of hsc70 ATPase domain mutants on endocytosis and recycling were specific, we determined the effects of overexpression of hsc70WT or hsc70K71M, the most potent mutant, on vesicular trafficking along the secretory pathway. Cells were pulse labeled with [35S]methionine, and the delivery of newly synthesized TfnR from the ER to cis- and trans-Golgi compartments was monitored by acquisition of Endo H resistance and the addition of complex oligosaccharides, respectively. Quantitation of the results shown in Fig. 3 A reveal that Endo H–resistant (Fig. 3 B) and mature (Fig. 3 C) forms of TfnR appeared at similar rates in control and hsc70WT-expressing cells. The corresponding rates observed in hsc70K71M-expressing cells were slightly inhibited (approximately twofold). The rates of biosynthesis and translocation of TfnR into the ER, as assessed by radiolabel incorporation and addition of core oligosaccharides, were also not significantly inhibited with overexpression of hsc70K71M (data not shown). Thus, TfnR biosynthesis was only slightly inhibited by the most potent inhibitor of BXX-Tfn uptake and recycling.

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