Role of Cyclin G-associated Kinase in Uncoating Clathrin-coated Vesicles from Non-neuronal Cells*
- From the ‡Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-0301 and the§Osaka University, Research Institute for Microbial Diseases, Department of Molecular Genetics, 3-1 Yamadaoka, Suita, Osaka-565-0871, Japan
Abstract
Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Laboratory of Cell Biology, Bldg. 3, B1-22, National Institutes of Health, Bethesda, MD 20892-0301. Tel.: 301-469-1228; Fax: 301-402-1519; E-mail: greenel@helix.nih.gov.
- Abbreviations:
- Hsc70
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the constitutive isoform of the 70-kDa heat shock protein
- AP
-
assembly protein
- GAK
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cyclin G-associated kinase
- AUX
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auxilin
- mAb
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monoclonal antibodies
- MES
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2-(N-morpholino)ethanesulfonic acid
- PAGE
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polyacrylamide gel electorphoresis
- GST
-
glutathioneS-transferase
- PBS
-
phosphate-buffered saline
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- Received September 9, 1999.
- Revision received October 19, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
