title: Identification of the endocytic sorting signal recognized by the Art1-Rsp5 ubiquitin ligase complex.
abstract: Targeted endocytosis of plasma membrane (PM) proteins allows cells to adjust their complement of membrane proteins to changing extracellular conditions. For a wide variety of PM proteins, initiation of endocytosis is triggered by ubiquitination. In yeast, arrestin-related trafficking adaptors (ARTs) enable a single ubiquitin ligase, Rsp5, to specifically and selectively target a wide range of PM proteins for ubiquitination and endocytosis. However, the mechanisms that allow ARTs to specifically recognize their appropriate substrates are unknown. We present the molecular features in the methionine permease Mup1 that are required for Art1-Rsp5-mediated ubiquitination and endocytosis. A combination of genetics, fluorescence microscopy, and biochemistry reveals three critical features that comprise an ART sorting signal in the Mup1 N-terminal cytosolic tail: 1) an extended acidic patch, 2) in close proximity to the first Mup1 transmembrane domain, and 3) close to the ubiquitinated lysines. We show that a functionally similar ART sorting signal is also required for the endocytosis of a second Art1-dependent cargo, Can1, suggesting a common mechanism for recognition of Art1 substrates. We isolate two separate suppressor mutations in the Art1 C-terminal domain that allele-specifically restore endocytosis of two Mup1 acidic patch mutants, consistent with an interaction between the Art1 C-terminus and the Mup1 acidic patch. We propose that this interaction is required for recruitment of the Art1-Rsp5 ubiquitination complex.
authors: Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853.
authors: Evan L
authors: EL
authors: Guiney
authors: Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853.
authors: Till
authors: T
authors: Klecker
authors: Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853 sde26@cornell.edu.
authors: Scott D
authors: SD
authors: Emr
mesh_terms: D000595:Amino Acid Sequence; D019393:Arrestin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D021382:Protein Sorting Signals; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D019393:Arrestin; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011506:Proteins; C062214:SWI4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D025801:Ubiquitin; C035737:major urinary proteins; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae