Limits...
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.

Show MeSH

Related in: MedlinePlus

Overexpression of hsc70 mutants dominantly interferes with TfnR endocytosis and recycling. (A) Adenovirally infected tTA HeLa cells overexpressing hsc70 wild-type and mutant proteins were harvested 18 h after infection and analyzed by SDS-PAGE and Coomassie blue staining. The 70-kD hsc/hsp70 bands are shown. Western blotting with antibodies 3C5, SPA-810, and 12CA5 identified the hsc/hsp70 species as indicated. (B) Adenovirally infected tTA HeLa cells expressing wild type (•), D199S (♦), K71M (▴), and T204V (▪) hsc70 were assayed for a single round of BXX-Tfn uptake and recycling. Control cells (▵) were infected with hsc70K71M-encoding adenoviruses but were cultured in the presence of tetracycline to suppress expression. The inset expands the data collected within the initial 5 min. The error bars reflect the SD from three independent experiments. WT, wild type.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196005&req=5

Figure 2: Overexpression of hsc70 mutants dominantly interferes with TfnR endocytosis and recycling. (A) Adenovirally infected tTA HeLa cells overexpressing hsc70 wild-type and mutant proteins were harvested 18 h after infection and analyzed by SDS-PAGE and Coomassie blue staining. The 70-kD hsc/hsp70 bands are shown. Western blotting with antibodies 3C5, SPA-810, and 12CA5 identified the hsc/hsp70 species as indicated. (B) Adenovirally infected tTA HeLa cells expressing wild type (•), D199S (♦), K71M (▴), and T204V (▪) hsc70 were assayed for a single round of BXX-Tfn uptake and recycling. Control cells (▵) were infected with hsc70K71M-encoding adenoviruses but were cultured in the presence of tetracycline to suppress expression. The inset expands the data collected within the initial 5 min. The error bars reflect the SD from three independent experiments. WT, wild type.

Mentions: Hsc70 is abundant, constituting ∼1–2% of total cellular protein, and can be detected by Coomassie blue staining of cell lysates (Fig. 2 A, the identity of each species was confirmed by Western blotting). To generate robust hsc70 ATPase mutant expression, we used a tetracycline-regulated adenoviral expression system. The cDNA encoding the wild-type and mutant hsc70 proteins included a sequence encoding an NH2-terminal HA tag in order to distinguish the overexpressed protein from the endogenous protein. Control experiments established that the HA tag did not significantly affect the properties of recombinantly generated hsc70 (data not shown). Reasonable overexpression (approximately threefold) of the mutant protein relative to endogenous hsc70 was attained at high viral levels, 400 pfu/cell (Fig. 2 A). Interestingly, some induction of hsp70, the inducible form of hsc70, was seen in cells expressing hsc70K71M and hsc70T204V. Within these cells, the level of hsp70 equaled that of endogenous hsc70, suggesting that overexpression of inhibitory hsc70 mutants might stress the cell. Under these conditions, however, infected cells exhibited normal morphology as observed by light microscopy and EM analysis (online supplemental Figure S1, available at http://www.jcb.org/cgi/content/full/152/3/607/DC1). If higher viral concentrations were used with expression of the mutant proteins, cells would round up and detach. Potential aberrant effects arising from the use of high virus levels were controlled by assaying a sample infected with hsc70K71M virus in the presence of tetracycline to repress expression of the adenovirally introduced gene (Fig. 2 A).


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 hsc70 mutants dominantly interferes with TfnR endocytosis and recycling. (A) Adenovirally infected tTA HeLa cells overexpressing hsc70 wild-type and mutant proteins were harvested 18 h after infection and analyzed by SDS-PAGE and Coomassie blue staining. The 70-kD hsc/hsp70 bands are shown. Western blotting with antibodies 3C5, SPA-810, and 12CA5 identified the hsc/hsp70 species as indicated. (B) Adenovirally infected tTA HeLa cells expressing wild type (•), D199S (♦), K71M (▴), and T204V (▪) hsc70 were assayed for a single round of BXX-Tfn uptake and recycling. Control cells (▵) were infected with hsc70K71M-encoding adenoviruses but were cultured in the presence of tetracycline to suppress expression. The inset expands the data collected within the initial 5 min. The error bars reflect the SD from three independent experiments. WT, wild type.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Overexpression of hsc70 mutants dominantly interferes with TfnR endocytosis and recycling. (A) Adenovirally infected tTA HeLa cells overexpressing hsc70 wild-type and mutant proteins were harvested 18 h after infection and analyzed by SDS-PAGE and Coomassie blue staining. The 70-kD hsc/hsp70 bands are shown. Western blotting with antibodies 3C5, SPA-810, and 12CA5 identified the hsc/hsp70 species as indicated. (B) Adenovirally infected tTA HeLa cells expressing wild type (•), D199S (♦), K71M (▴), and T204V (▪) hsc70 were assayed for a single round of BXX-Tfn uptake and recycling. Control cells (▵) were infected with hsc70K71M-encoding adenoviruses but were cultured in the presence of tetracycline to suppress expression. The inset expands the data collected within the initial 5 min. The error bars reflect the SD from three independent experiments. WT, wild type.
Mentions: Hsc70 is abundant, constituting ∼1–2% of total cellular protein, and can be detected by Coomassie blue staining of cell lysates (Fig. 2 A, the identity of each species was confirmed by Western blotting). To generate robust hsc70 ATPase mutant expression, we used a tetracycline-regulated adenoviral expression system. The cDNA encoding the wild-type and mutant hsc70 proteins included a sequence encoding an NH2-terminal HA tag in order to distinguish the overexpressed protein from the endogenous protein. Control experiments established that the HA tag did not significantly affect the properties of recombinantly generated hsc70 (data not shown). Reasonable overexpression (approximately threefold) of the mutant protein relative to endogenous hsc70 was attained at high viral levels, 400 pfu/cell (Fig. 2 A). Interestingly, some induction of hsp70, the inducible form of hsc70, was seen in cells expressing hsc70K71M and hsc70T204V. Within these cells, the level of hsp70 equaled that of endogenous hsc70, suggesting that overexpression of inhibitory hsc70 mutants might stress the cell. Under these conditions, however, infected cells exhibited normal morphology as observed by light microscopy and EM analysis (online supplemental Figure S1, available at http://www.jcb.org/cgi/content/full/152/3/607/DC1). If higher viral concentrations were used with expression of the mutant proteins, cells would round up and detach. Potential aberrant effects arising from the use of high virus levels were controlled by assaying a sample infected with hsc70K71M virus in the presence of tetracycline to repress expression of the adenovirally introduced gene (Fig. 2 A).

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