title: Evidence that the 50-kDa substrate of brefeldin A-dependent ADP-ribosylation binds GTP and is modulated by the G-protein beta gamma subunit complex.
abstract: Brefeldin A, a fungal metabolite that inhibits membrane transport, induces the mono(ADP-ribosyl)ation of two cytosolic proteins of 38 and 50 kDa as judged by SDS/PAGE. The 38-kDa substrate has been previously identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We report that the 50-kDa BFA-induced ADP-ribosylated substrate (BARS-50) has native forms of 170 and 130 kDa, as determined by gel filtration of rat brain cytosol, indicating that BARS-50 might exist as a multimeric complex. BARS-50 can bind GTP, as indicated by blot-overlay studies with [alpha-32P]GTP and by photoaffinity labeling with guanosine 5'-[gamma-32P] [beta,gamma-(4-azidoanilido)]triphosphate. Moreover, ADP-ribosylation of BARS-50 was completely inhibited by the beta gamma subunit complex of G proteins, while the ADP-ribosylation of GAPDH was unmodified, indicating that this effect was due to an interaction of the beta gamma complex with BARS-50, rather than with the ADP-ribosylating enzyme. Two-dimensional gel electrophoresis and immunoblot analysis shows that BARS-50 is a group of closely related proteins that appear to be different from all the known GTP-binding proteins.
authors.affiliation: Laboratory of Cellular and Molecular Endocrinology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Di Girolamo
authors.forename: M G
authors.initials: MG
authors.lastname: Silletta
authors.forename: M A
authors.initials: MA
authors.lastname: De Matteis
authors.forename: A
authors.initials: A
authors.lastname: Braca
authors.forename: A
authors.initials: A
authors.lastname: Colanzi
authors.forename: D
authors.initials: D
authors.lastname: Pawlak
authors.forename: M M
authors.initials: MM
authors.lastname: Rasenick
authors.forename: A
authors.initials: A
authors.lastname: Luini
authors.forename: D
authors.initials: D
authors.lastname: Corda
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000096:biosynthesis*; D000345:Affinity Labels; D000818:Animals; D001921:Brain / Q000378:metabolism; D020126:Brefeldin A; D002417:Cattle; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D003517:Cyclopentanes; D006150:Guanine Nucleotides; D008565:Membrane Proteins; D020126:Brefeldin A; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins