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PMID: 7624370 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 92 ·No. 15 ·1995-07-18 ·Pages 7065-9

Di Girolamo M, Silletta MG, De Matteis MA, Braca A, Colanzi A, Pawlak D, Rasenick MM, Luini A, Corda D

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.

MeSH Terms
Adenosine Diphosphate Ribose/biosynthesis Affinity Labels Animals Brain/metabolism Brefeldin A Cattle Cells, Cultured Cyclopentanes/metabolism Cytosol/metabolism Electrophoresis, Gel, Two-Dimensional GTP-Binding Proteins/metabolism Guanine Nucleotides/metabolism Guanosine Triphosphate/metabolism Membrane Proteins/metabolism Protein Binding Protein Conformation Protein Processing, Post-Translational Rats Thyroid Gland/cytology,metabolism
Chemicals
Affinity Labels Cyclopentanes Guanine Nucleotides Membrane Proteins Brefeldin A Adenosine Diphosphate Ribose Guanosine Triphosphate GTP-Binding Proteins
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Di Girolamo 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
De Matteis M A
Braca A
Colanzi A
Pawlak D
Rasenick M M
Luini A
Corda D
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.92.15.7065
Published
1995-07-18
Pages
7065-9
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC41472
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.92.15.7065 PMC Full Text View journal details
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