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.
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.
Di Girolamo
M
M
Laboratory of Cellular and Molecular Endocrinology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy.
Silletta
M G
MG
De Matteis
M A
MA
Braca
A
A
Colanzi
A
A
Pawlak
D
D
Rasenick
M M
MM
Luini
A
A
Corda
D
D
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
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