Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics.
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, gamma-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.
Wasiak
Sylwia
S
Department of Neurology and Neurosurgery, Montreal Neurological Institute, Montreal Quebec H3A 2B4, Canada.
Legendre-Guillemin
Valerie
V
Puertollano
Rosa
R
Blondeau
Francois
F
Girard
Martine
M
de Heuvel
Elaine
E
Boismenu
Daniel
D
Bell
Alexander W
AW
Bonifacino
Juan S
JS
McPherson
Peter S
PS
D033961:Adaptor Protein Complex 1 / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D040901:Proteomics; D051381:Rats
D033961:Adaptor Protein Complex 1; D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D002966:Clathrin; C405944:GGA2 protein, human; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins