Identification of residues in TIP47 essential for Rab9 binding.
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.
Hanna
John
J
Department of Biochemistry, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305-5307, USA.
Carroll
Kate
K
Pfeffer
Suzanne R
SR
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
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