Guanine nucleotide-dependent ADP-ribosylation of soluble rho catalyzed by Clostridium botulinum C3 ADP-ribosyltransferase. Isolation and characterization of a newly recognized form of rhoA.
Two C3 ADP-ribosyltransferase substrates with different characteristics were isolated from bovine brain cytosol. Amino acid sequences of tryptic peptides from the two substrates were identical to rhoA and rhoB; hence, the purified proteins are referred to as rhoA* and rhoB*, respectively. Soluble rhoA* exhibits properties different from those previously reported for rho proteins. In contrast to other C3 substrates, rhoA* behaved as a 77-80-kDa protein on gel filtration, although on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the ADP-ribosylated moiety had a mobility consistent with a 21.5-kDa protein. Furthermore, C3-catalyzed ADP-ribosylation of rhoA* was dependent on guanine nucleotides in the presence of 1 mM Mg2+ or 1 mM EDTA (0.19 microM free Mg2+). Half-maximal stimulation by GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), guanylyl-imidodiphosphate (Gpp(NH)p), and GDP was observed at 16, 20, 220, and 380 nM, respectively; guanosine 5'-O-(2-thiodiphosphate), GMP, and adenine nucleotides were ineffective. In the presence of GTP gamma S, the rate and extent of ADP-ribosylation was enhanced by dimyristoylphosphatidylcholine and/or cholate. This increase in ADP-ribosylation was specific for rhoA*; it was not observed with rhoB* and has not been reported for other C3 substrates. These distinct properties suggest that rhoA* is a newly recognized type of C3 substrate, differing from the rhoA-like proteins previously reported. rhoB*, on the other hand, has properties similar to those reported for membrane-associated rhoB and its ADP-ribosylation was independent of guanine nucleotides in the presence of 1 mM Mg2+ and not affected by dimyristoylphosphatidylcholine and/or cholate.
Williamson
K C
KC
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Smith
L A
LA
Moss
J
J
Vaughan
M
M
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001905:Botulinum Toxins; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003014:Clostridium botulinum / Q000201:enzymology*; D004134:Dimyristoylphosphatidylcholine / Q000494:pharmacology; D017886:Durapatite; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006882:Hydroxyapatites; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D020743:rhoB GTP-Binding Protein
D002793:Cholic Acids; D006882:Hydroxyapatites; D008565:Membrane Proteins; D010446:Peptide Fragments; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000244:Adenosine Diphosphate; D017886:Durapatite; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020743:rhoB GTP-Binding Protein; D019826:Cholic Acid; D004134:Dimyristoylphosphatidylcholine