title: The GTP-bound and Sumoylated Form of the rab17 Small Molecular Weight GTPase Selectively Binds Syntaxin 2 in Polarized Hepatic WIF-B Cells.
abstract: A major focus for our laboratory is identifying the molecules and mechanisms that regulate polarized apical protein sorting in hepatocytes, the major epithelial cells of the liver. These trafficking pathways are regulated, in part, by small molecular weight rab GTPases. We chose to investigate rab17, whose expression is restricted to polarized epithelial cells, is enriched in liver, and has been implicated in regulating basolateral to apical transcytosis. To initiate our studies, we generated three recombinant adenoviruses expressing wild type, constitutively active (GTP bound), or dominant-negative (GDP bound) rab17. Immunoblotting revealed rab17 immunoreactive species at 25 kDa (the predicted rab17 molecular mass) and 40 kDa. We determined that mono-sumoylation of the 25-kDa rab17 is responsible for the shift in molecular mass, and that rab17 prenylation is required for sumoylation. We further determined that sumoylation selectively promotes interactions with syntaxin 2 (but not syntaxins 3 or 4) and that these interactions are nucleotide dependent. Furthermore, a K68R-mutated rab17 led to the redistribution of syntaxin 2 and 5' nucleotidase from the apical membrane to subapical puncta, whereas multidrug resistance protein 2 distributions were not changed. Together these data are consistent with the proposed role of rab17 in vesicle fusion with the apical plasma membrane and further implicate sumoylation as an important mediator of protein-protein interactions. The selectivity in syntaxin binding and apical protein redistribution further suggests that rab17 and syntaxin 2 mediate fusion of transcytotic vesicles at the apical surface.
authors.affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.
authors.forename: Anneliese C
authors.initials: AC
authors.lastname: Striz
authors.affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064 tuma@cua.edu.
authors.forename: Pamela L
authors.initials: PL
authors.lastname: Tuma
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002460:Cell Line; D008099:Liver / Q000166:cytology / Q000276:immunology*; D051379:Mice; D020125:Mutation, Missense; D058207:Sumoylation / Q000235:genetics / Q000276:immunology*; D050827:Syntaxin 1 / Q000235:genetics / Q000276:immunology*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D050827:Syntaxin 1; C000605623:Rab17 protein, mouse; D020691:rab GTP-Binding Proteins
keywords: Rab; SNARE proteins; hepatocyte; small GTPase; sumoylation