title: Identification of residues in TIP47 essential for Rab9 binding.
abstract: TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-dependent and cation-independent mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the trans-Golgi network in vitro and in living cells. TIP47 recognizes distinct determinants in the cytoplasmic domains of these two receptors, and its ability to bind to the cation-independent MPR is enhanced by the concomitant binding of the Rab9 GTPase. We show here that TIP47 residues 161-169 are essential, but likely not sufficient, for Rab9 binding. Mutation of these residues led to a significant decrease in Rab9 binding, but did not alter the global folding of the protein. The most impaired mutant was indistinguishable from wild-type TIP47 in its circular dichroism spectrum, and mutant proteins that showed decreased Rab9 binding retained full capacity to bind to MPR cytoplasmic domains. Closely related sequences in a related protein, adipophilin, did not confer Rab9 binding capacity to that protein. Partial proteolysis of TIP47 and TIP47 mutant proteins revealed subtle conformational differences, suggesting that residues 161-169 reside in a portion of TIP47 that is important for its conformation. These experiments reveal distinct binding domains for the Rab9 GTPase and MPR cytoplasmic domains in the cargo selection protein TIP47.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305-5307, USA.
authors.forename: John
authors.initials: J
authors.lastname: Hanna
authors.forename: Kate
authors.initials: K
authors.lastname: Carroll
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Pfeffer
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D000070780:Perilipin-2; D000070796:Perilipin-3; D011257:Pregnancy Proteins; D017510:Protein Folding; D021381:Protein Transport; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C000602982:PLIN2 protein, human; C038196:PLIN3 protein, human; D010446:Peptide Fragments; D010455:Peptides; D000070780:Perilipin-2; D000070796:Perilipin-3; D011257:Pregnancy Proteins; D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; C079856:RAB9A protein, human; D020691:rab GTP-Binding Proteins