Distinct roles for TGN/endosome epsin-like adaptors Ent3p and Ent5p.
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 alpha-factor maturation defects were observed when ent5Delta but not ent3Delta was introduced together with deletions of the GGA genes. In AP-1-deficient cells, ent3Delta and to a lesser extent ent5Delta caused minor alpha-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.
Costaguta
Giancarlo
G
Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
Duncan
Mara C
MC
Fernández
G Esteban
GE
Huang
Grace H
GH
Payne
Gregory S
GS
D020727:ADP-Ribosylation Factors / Q000172:deficiency; D033961:Adaptor Protein Complex 1 / Q000172:deficiency; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D000483:Alleles; D000595:Amino Acid Sequence; D011992:Endosomes / Q000378:metabolism*; D017353:Gene Deletion; D008954:Models, Biological; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D021601:trans-Golgi Network / Q000378:metabolism*
D033961:Adaptor Protein Complex 1; D033942:Adaptor Proteins, Vesicular Transport; C495587:Ent3 protein, S cerevisiae; C495588:Ent5 protein, S cerevisiae; C407306:GGA adaptor proteins; C497361:Gga2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C114154:epsin; D020727:ADP-Ribosylation Factors