Molecular cloning and functional characterization of brefeldin A-ADP-ribosylated substrate. A novel protein involved in the maintenance of the Golgi structure.
Brefeldin A (BFA) is a fungal metabolite that disassembles the Golgi apparatus into tubular networks and causes the dissociation of coatomer proteins from Golgi membranes. We have previously shown that an additional effect of BFA is to stimulate the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa (brefeldin A-ADP-riboslyated substrate (BARS)) and that this effect greatly facilitates the Golgi-disassembling activity of the toxin. In this study, BARS has been purified from rat brain cytosol and microsequenced, and the BARS cDNA has been cloned. BARS shares high homology with two known proteins, C-terminal-binding protein 1 (CtBP1) and CtBP2. It is therefore a third member of the CtBP family. The role of BARS in Golgi disassembly by BFA was verified in permeabilized cells. In the presence of dialyzed cytosol that had been previously depleted of BARS or treated with an anti-BARS antibody, BFA potently disassembled the Golgi. However, in cytosol complemented with purified BARS, or even in control cytosols containing physiological levels of BARS, the action of BFA on Golgi disassembly was strongly inhibited. These results suggest that BARS exerts a negative control on Golgi tubulation, with important consequences for the structure and function of the Golgi complex.
Spanò
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S
Istituto di Ricerche Farmacologiche "Mario Negri", Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), 20157 Milano, Italy.
Silletta
M G
MG
Colanzi
A
A
Alberti
S
S
Fiucci
G
G
Valente
C
C
Fusella
A
A
Salmona
M
M
Mironov
A
A
Luini
A
A
Corda
D
D
Spanfò
S
S
D020727:ADP-Ribosylation Factors; D000429:Alcohol Oxidoreductases; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology*; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects*; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D013154:Spleen / Q000378:metabolism; D014157:Transcription Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; C119945:Ctbp1 protein, rat; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D014157:Transcription Factors; D020126:Brefeldin A; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors