Extensive accumulation of an extracellular l‐amino‐acid oxidase during gametogenesis of Chlamydomonas reinhardtii

Abstract

In a previous study [BultC, L. & Wollman, F.-A. (1992) Eur: J. Biochem. 204, 327-3361, we identified a novel gamete-specific polypeptide of Chlamydomonas reinhardtii, Ma. This 66-kDa polypeptide reacts with antibodies to cytochrome f and accumulates in gametes only in conditions that promote destabilisation of the cytochrome bJf complex. Here, we show that Ma is not a modification product of cytochrome J but is part of protein M, a high-molecular-mass L-amino-acid oxidase located in the periplasm. It catalyzes oxidation of all L-amino acids tested, except cysteine. Using phenylalanine as a substrate, saturation of the enzymatic rate is reached at 2 pM. These characteristics suggest that protein M may operate in vivo as an efficient scavanger of ammonium from extracellular amino acids. The enzyme contains non-covalently bound FAD. It exists in two forms with essentially similar enzymatic properties, of 1.2-1.3 MDa and 0.9-1 . O MDa, respectively. The lighter form is an oligomer of Ma, while the heavier form contains, in addition to Ma, a second polypeptide of 135 kDa, Mp, in a molar ratio of 3 -4 MaMp. Both polypeptides are glycosylated.In a previous study Wollman, 1990, 1992), we have shown that cytochrome bdf is specifically removed from the thylakoid membrane during gametogenesis of Chlamydomonas reinhardtii, resulting in an inactivation of photosynthesis in aging gametes. In parallel with the removal of the subunits of the cytochrome bJf complex, we observed a continuous accumulation of a gamete-specific protein, which we called Ma, showing immunological cross-reactivity with cytochromef. This 66-kDa polypeptide, becomes one of the major polypeptides in gametes deprived of nitrogen for more than 50 h. The production of Ma and the disappearance of cytochrome bJf occurred only in conditions that promote starch accumulation during gametogenesis, i.e. they were prevented when acetate was omitted from the medium, or when mitochondrial respiration was impaired by mutations or inhibitors. Developmental control was also operating, since neither phenomenon was observed in nitrogen-starved cultures of mutant C4, which has a block in gametic differentiation (Bultt and Bennoun, 1990).In the present paper, we have further characterized the biochemical properties and the function of polypeptide Ma. In particular we have shown that the immunological crossreaction between cytochrome f and Ma is due to a short shared protein motif and we demonstrate that Ma is the major subunit of protein M, a gamete-specific L-amino-acid oxidase located in the periplasm. MATERIALS AND METHODS Strains and culture conditionsVegetative cells of the '137c' wild type and CW15 (Davies and Plaskitt, 1971) strains of C. reinhardtii were grown on Tris/acetate/phosphate medium (Harris, 1989) at 28 OC, under 500-1x continuous illumination. Gametogenesis was triggered by diluting 1 vol. of a vegetative culture in stationary phase (2 X 10' cells/ml) into 7-10 vol. of the same medium from which all sources of nitrogen were omitted (NO medium). Flasks ...

L-Amino Acid Oxidases with Specificity for Basic L-Amino Acids in Cyanobacteria

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Gametogenesis in the Chlamydomonas reinhardtii minus Mating Type Is Controlled by Two Genes, MID and MTD1

“…An onset of massive protein and nucleic acid catabolism (Beck and Haring 1996) and ribosome degradation (Martin et al 1976), accompanied by an early (within 1-3 hr) expression of genes encoding proteasome subunits (Abe et al 2004), amino acid oxidase (Vallon et al 1993), urate oxidase (Merchan et al 2001), and other proteins presumably involved in this catabolic response (Abe et al 2004). The outcome of this is that cells enter a stable G 0 stage in which they can survive for weeks without an exogenous nitrogen source.…”

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