title: Requirements for recruitment of a G protein-coupled receptor to clathrin-coated pits in budding yeast.
abstract: Endocytic internalization of G protein-coupled receptors (GPCRs) plays a critical role in down-regulation of GPCR signaling. The yeast mating pheromone receptor Ste2p has been used as a model to investigate mechanisms of signal transduction, modification, and endocytic internalization of GPCRs. We previously used a fluorescently labeled mating pheromone derivative to reveal unappreciated molecular and spatiotemporal features of GPCR endocytosis in budding yeast. Here, we identify recruitment of Ste2p to preexisting clathrin-coated pits (CCPs) as a key step regulated by receptor phosphorylation and subsequent ubiquitination upon ligand binding. The yeast casein kinase I homologue Yck2p directly phosphorylates six serine residues located in the C-terminal tail of Ste2p, and mutation of these serine residues to alanine significantly decreased recruitment of Ste2p to CCPs. We also found that the clathrin adaptors Ent1p, Ent2p, and Ede1p work cooperatively to recruit ubiquitinated Ste2p to CCPs. In addition, ubiquitination has a role in ligand-independent constitutive recruitment of Ste2p to CCPs, although this process is much slower than ligand-induced recruitment. These results suggest that ubiquitination of Ste2p is indispensable for recruiting Ste2p to CCPs in both ligand-dependent and ligand-independent endocytosis.
authors.affiliation: Department of Biological Science and Technology, Tokyo University of Science, Yamazaki 2641, Noda, Chiba 278-8510, Japan.
authors.forename: Junko Y
authors.initials: JY
authors.lastname: Toshima
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Nakanishi
authors.forename: Kensaku
authors.initials: K
authors.lastname: Mizuno
authors.forename: Jiro
authors.initials: J
authors.lastname: Toshima
authors.forename: David G
authors.initials: DG
authors.lastname: Drubin
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004705:Endocytosis; D008024:Ligands; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D057149:Ubiquitinated Proteins / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D002966:Clathrin; D008024:Ligands; C048632:MF(ALPHA)1 protein, S cerevisiae; D011498:Protein Precursors; D043562:Receptors, G-Protein-Coupled; D029701:Saccharomyces cerevisiae Proteins; D057149:Ubiquitinated Proteins