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Vol. 17, Issue 9, 3907-3920, September 2006

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Distinct Roles for TGN/Endosome Epsin-like Adaptors Ent3p and Ent5p

Giancarlo Costaguta^*, Mara C. Duncan^*, G. Esteban Fernández^*, Grace H. Huang, and Gregory S. Payne^*

*Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095; and Department of Biostatistics, School of Public Health, UCLA, Los Angeles, CA 90095

Submitted May 11, 2006; Revised June 12, 2006; Accepted June 14, 2006
Monitoring Editor: Janet Shaw

Clathrin adaptors are key factors in clathrin-coated vesicle formation, coupling clathrin to cargo and/or the lipid bilayer. A physically interacting network of three classes of adaptors participate in clathrin-mediated traffic between the trans-Golgi network (TGN) and endosomes: AP-1, Gga proteins, and epsin-like proteins. Here we investigate functional relationships within this network through transport assays and protein localization analysis in living yeast cells. We observed that epsin-like protein Ent3p preferentially localized with Gga2p, whereas Ent5p distributed equally between AP-1 and Gga2p. Ent3p was mislocalized in Gga-deficient but not in AP-1–deficient cells. In contrast, Ent5p retained localization in cells lacking either or both AP-1 and Gga proteins. The Ent proteins were dispensable for AP-1 or Gga localization. Synthetic genetic growth and -factor maturation defects were observed when ent5 but not ent3 was introduced together with deletions of the GGA genes. In AP-1–deficient cells, ent3 and to a lesser extent ent5 caused minor -factor maturation defects, but together resulted in a near-lethal phenotype. Deletions of ENT3 and ENT5 also displayed synthetic defects similar to, but less severe than, synthetic effects of AP-1 and Gga inactivation. These results differentiate Ent3p and Ent5p function in vivo, suggesting that Ent3p acts primarily with Gga proteins, whereas Ent5p acts with both AP-1 and Gga proteins but is more critical for AP-1–mediated transport. The data also support a model in which the Ent adaptors provide important accessory functions to AP-1 and Gga proteins in TGN/endosome traffic.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-05-0410) on June 21, 2006.

Address correspondence to: Gregory S. Payne (gpayne{at}mednet.ucla.edu)

Abbreviations used: CCV, clathrin-coated vesicle; TGN, trans-Golgi network; CPS, carboxypeptidase S; ALP, alkaline phosphatase; ts, thermosensitive; ENTH, epsin N-terminal homology; ANTH, AP180 N-terminal homology; mRFP, monomeric red fluorescent protein; GFP, green fluorescent protein.

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