Characterization of Chlamydomonas 102 and 104 Mutants Reveals Intermolecular Complementation in the Molybdenum Cofactor Protein CNX1E

Abstract

Chlamydomonas reinhardtii is a eukaryotic, unicellular, biflagellate green alga. In Chlamydomonas nitrate assimilation depends on the activity of the enzyme nitrate reductase that requires the molybdenum cofactor (Moco). We have characterized the Chlamydomonas strains 102 (nit5nit6), 104 (nit4nit5) and 106 (nit6). These mutations lead to the exchanges V171A (nit4) and G183D (nit6) in the CNX1E protein of Moco biosynthesis that result in Moco deficiency and hence inability to grow on nitrate. CNX1E inserts molybdenum into molybdopterin (MPT) to yield Moco through the intermediate MPT-AMP. The CNX1E variant G183D is unable to bind MPT-AMP. However, the CNX1E variant V171A is able to bind MPT-AMP but is not able to hydrolyze it and insert the molybdenum to obtain Moco. We show that intermolecular complementation between the CNX1E variants V171A and G183D both in vivo and in vitro restored its activity. The molecular characterization of strain 106 led us to discover the presence of wild type and nit6 CNX1E alleles in this strain (nit6/+). Then, we found out that 106 is indeed a diploid strain. It is proposed that 102, the strain from which 106 derived, may contain a genetic alteration that produces an aberrant meiotic division of the mature zygotes.