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Sylwia Wasiak¹, Valerie Legendre-Guillemin¹, Rosa Puertollano³, Francois Blondeau¹, Martine Girard¹, Elaine de Heuvel¹, Daniel Boismenu², Alexander W. Bell², Juan S. Bonifacino³ and Peter S. McPherson¹

¹ Department of Neurology and Neurosurgery, Montreal Neurological Institute
² Montreal Proteomics Centre, McGill University, Montreal Quebec H3A 2B4, Canada
³ Cell Biology and Metabolism Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892

Address correspondence to Peter S. McPherson, CBET Group, Dept. of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, 3801 University St., Montreal, Quebec H3A 2B4, Canada. Tel.: (514) 398-7355. Fax: (514) 398-8106. E-mail: peter.mcpherson{at}mcgill.ca

Despite numerous advances in the identification of the molecular machinery for clathrin-mediated budding at the plasma membrane, the mechanistic details of this process remain incomplete. Moreover, relatively little is known regarding the regulation of clathrin-mediated budding at other membrane systems. To address these issues, we have utilized the powerful new approach of subcellular proteomics to identify novel proteins present on highly enriched clathrin-coated vesicles (CCVs). Among the ten novel proteins identified is the rat homologue of a predicted gene product from human, mouse, and Drosophila genomics projects, which we named enthoprotin. Enthoprotin is highly enriched on CCVs isolated from rat brain and liver extracts. In cells, enthoprotin demonstrates a punctate staining pattern that is concentrated in a perinuclear compartment where it colocalizes with clathrin and the clathrin adaptor protein (AP)1. Enthoprotin interacts with the clathrin adaptors AP1 and with Golgi-localized, -ear–containing, Arf-binding protein 2. Through its COOH-terminal domain, enthoprotin binds to the terminal domain of the clathrin heavy chain and stimulates clathrin assembly. These data suggest a role for enthoprotin in clathrin-mediated budding on internal membranes. Our study reveals the utility of proteomics in the identification of novel vesicle trafficking proteins.

Key Words: clathrin adaptors; endosome; ENTH domain; GGAs; TGN

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