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PMID:16687570

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Citation

Xiao, J, Kim, LS and Graham, TR (2006) Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo. Mol. Biol. Cell 17:3281-90

Abstract

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.

Links

PubMed PMC1483056 Online version:10.1091/mbc.E06-02-0106

Keywords

Amino Acid Motifs; Auxilins/metabolism; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; Clathrin/metabolism; HSP70 Heat-Shock Proteins/metabolism; Molecular Chaperones/chemistry; Molecular Chaperones/metabolism; Phosphoproteins/chemistry; Phosphoproteins/genetics; Phosphoproteins/metabolism; Protein Structure, Tertiary/genetics; Repetitive Sequences, Amino Acid/genetics; Saccharomyces cerevisiae/cytology; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/chemistry; Saccharomyces cerevisiae Proteins/genetics; Saccharomyces cerevisiae Proteins/metabolism; Sequence Deletion; Vesicular Transport Proteins

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

YEAST:SWA2

enables

GO:0030276: clathrin binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:SWA2

involved_in

GO:0032781: positive regulation of ATP-dependent activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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