LOCATION OF CYSTEINE AND CYSTINE RESIDUES IN S-RIBONUCLEASES ASSOCIATED WITH GAMETOPHYTIC SELF-INCOMPATIBILITY

S-Ribonucleases (S-RNases) that cosegregate with S-alleles in the styl es of solanaceous and rosaceous plants are associated with gametophyti c self-incompatibility (GSI). The amino acid sequences of many S-RNase s have been derived from cDNA sequences, but the state of half-cystine s has not been clarified. We report the locations of the two free cyst eine residues and four disulfide bridges of tobacco S-6-RNase and of t he four disulfide bridges of Japanese pear S-4-RNase. The protein was first S-pyridylethylated at a low pH to selectively modify the free cy steines without thiol-disulfide exchange. The S-pyridylethylated prote in (PE-protein) was digested with Achromobacter protease I (API) at pH 6.5 then analyzed by liquid chromatography/electrospray-ionization ma ss spectrometry (LC/ESI-MS). This analysis showed that tobacco S-6-RNa se has two free cysteine residues, Cys77 and Cys95, and four disulfide bonds at Cys16-Cys21, Cys45-Cys94, Cys153-Cys182 and Cys165-Cys176. S imilarly, four disulfide bonds were identified for pear S-4-RNase, whi ch bears no free cysteine, at Cys15-Cys22, Cys48-Cys92, Cys156-Cys195 and Cys172-Cys183. The eight cysteines forming these four disulfide bo nds are conserved in all the known S-RNases, indicative that these cro ss-links are important in stabilizing the tertiary structures of the s elf-incompatibility-associated glycoproteins in the solanaceous and ro saceous families. The LC/ESI-MS analysis also provided some structural informations regarding sugar chains attached to the S-RNases.