A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis.
Sequencing of the entire genome of S. cerevisiae has revealed the existence of five proteins containing EH domains. These are protein-protein interaction modules first described in mammalian Eps15, a protein that is involved in clathrin-dependent endocytosis. Two of the yeast proteins, End3p and Pan1p, are required for the internalization step of endocytosis. We report characterization of the nonessential ORF YBL047c which, like Eps15, encodes a protein with three N-terminal EH domains. Deletion of YBL047c leads to a defective fluid-phase endocytosis and to defective internalization of the pheromone (alpha)-factor and uracil permease. We therefore named YBL047c EDE1, for EH Domains and Endocytosis. Ede1p expressed as a chromosomally encoded fusion to the green fluorescent protein is localized in punctate cortical spots that only partially colocalize with actin patches. This localization is maintained when actin is depolymerized. Deletion of EDE1 impairs the diploid budding pattern, but has only a small impact on actin cytoskeleton organization, in contrast to the effects observed in pan1 cells and many end mutants impaired in proteins colocalizing with cortical actin patches. Genetic interaction was observed between EDE1 and RSP5, which encodes the ubiquitin ligase Rsp5p essential for ubiquitin-dependent endocytosis of many plasma membrane proteins, thus further emphasizing the functional link between Rsp5p and the EH domain proteins. We also observed genetic interaction between EDE1, and END3 or PAN1, suggesting that Ede1p might be part of a yeast EH network implicated in endocytosis.
Gagny
B
B
Institut Jacques Monod, CNRS/Universités Paris VI et VII, 75251 Paris cedex 05, France.
Wiederkehr
A
A
Dumoulin
P
P
Winsor
B
B
Riezman
H
H
Haguenauer-Tsapis
R
R
D000199:Actins / Q000378:metabolism; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton; D004705:Endocytosis / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins; D011108:Polymers; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes
D000199:Actins; D003598:Cytoskeletal Proteins; C091908:END3 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; C111870:PAN1 protein, S cerevisiae; D011108:Polymers; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae