INCREASED TRANSCRIPT LEVELS OF A METHIONINE SYNTHASE DURING ADHESION-INDUCED ACTIVATION OF CHLAMYDOMONAS-REINHARDTII GAMETES

Chlamydomonas gametes of opposite mating types interact through flagel lar adhesion molecules called agglutinins leading to a signal transduc tion cascade that induces cell wall loss and activation of mating stru ctures along with other cellular responses that ultimately result in z ygote formation. To identify molecules involved in these complex cellu lar events, we have employed subtractive and differential hybridizatio n with cDNA from mt + gametes activated for fertilization and non-sign aling, vegetative (non-gametic) cells. We identified 55 cDNA clones wh ose transcripts were regulated in activated gametes. Here we report th e molecular cloning and characterization of the complementary DNA (cDN A) for one clone whose transcripts in activated gametes were several-f old higher than in normal gametes. Regulation of the transcript was no t related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins. The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa. Database search analysis transfer ase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine. This enzyme catalyzes transfer of a methy l group from 5-methyltetrahydrofolate to homocysteine resulting in met hionine formation. Affinity-purified polyclonal antibodies raised agai nst a bacterially produced GST-fusion protein identified a 85 kDa solu ble protein in Chlamydomonas gametes. Southern blot hybridization indi cated that the enzyme is encoded by a single-copy gene. The evidence p resented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization.