Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site.
Pertussis toxin catalyzes the transfer of ADP-ribose from NAD to the guanine nucleotide-binding regulatory proteins Gi, Go, and transducin. Based on a partial amino acid sequence for a tryptic peptide of ADP-ribosylated transducin, asparagine had been characterized as the site of pertussis toxin-catalyzed ADP-ribosylation. Subsequently, cDNA data for the alpha subunit of transducin indicated that the putative asparagine residue was, in fact, not present in the protein. To determine the amino acid that served as the ADP-ribose acceptor, radiolabel from [adenine-U-14C]NAD was incorporated, in the presence of pertussis toxin, into the alpha subunit of transducin (0.3 mol/mol). An ADP-ribosylated, tryptic peptide was purified and fully sequenced by automated Edman degradation. The amino acid sequence, Glu-Asn 343-Leu-Lys-Asp 346-X-Gly 348-Leu-Phe, corresponds to the cDNA sequence coding the carboxyl-terminal nonapeptide, Glu 342-Phe 350, which includes by cDNA sequence cysteine at position 347. Neither Asn 343 nor Asp 346 appeared to be modified; residue 347 adhered to the sequencing resin. Cysteine, the missing residue, was eluted from the sequencing resin with acetic acid along with 76% of the peptide-associated radioactivity, half of which, presumably ADP-ribosylcysteine, eluted from an anion exchange column between NAD and ADP-ribose; the other half had a retention time corresponding to 5'-AMP. We conclude that Cys 347 and not Asn 343 or Asp 346 is the site of pertusis toxin-catalyzed ADP-ribosylation in transducin.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D037342:Pertussis Toxin; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015293:Transducin; D014357:Trypsin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D000596:Amino Acids; D008565:Membrane Proteins; D009702:Nucleoside Diphosphate Sugars; D010446:Peptide Fragments; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D014357:Trypsin; D015293:Transducin; D003545:Cysteine