Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes.
Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation. To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells. We identified 55 cDNA clones whose transcripts were regulated in activated gametes. Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes. Regulation of the transcript was not 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 and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; 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 methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation. Affinity-purified polyclonal antibodies raised against a bacterially produced GST-fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes. Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene. The evidence presented 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.
Kurvari
V
V
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235-9039, USA.
Qian
F
F
Snell
W J
WJ
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli / Q000201:enzymology; D005306:Fertilization; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D005854:Germ Cells; D005982:Glutathione Transferase / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D005982:Glutathione Transferase