Spontaneous Mutations in the Ammonium Transport Gene AMT4 of Chlamydomonas reinhardtii

Abstract

Evidence in several microorganisms indicates that Amt proteins are gas channels for NH 3 and CH 3 NH 2 , and this has been confirmed structurally. Chlamydomonas reinhardtii has at least four AMT genes, the most reported for a microorganism. Under nitrogen-limiting conditions all AMT genes are transcribed and Chlamydomonas is sensitive to methylammonium toxicity. All 16 spontaneous methylammonium-resistant mutants that we analyzed had defects in accumulation of [ 14 C]methylammonium. Genetic crosses indicated that 12 had lesions in a single locus, whereas two each had lesions in other loci. Lesions in different loci were correlated with different degrees of defect in [ 14 C]methylammonium uptake. One mutant in the largest class had an insert in the AMT4 gene, and the insert cosegregated with methylammonium resistance in genetic crosses. The other 11 strains in this class also had amt4 lesions, which we characterized at the molecular level. Properties of the amt4 mutants were clearly different from those of rh1 RNAi lines. They indicated that the physiological substrates for Amt and Rh proteins, the only two members of their protein superfamily, are NH 3 and CO 2 , respectively.

CeRh1 ( rhr-1 ) is a dominant Rhesus gene essential for embryonic development and hypodermal function in Caenorhabditis elegans

“…Thus, during the evolutionary period of their co-occurrence, Rh and Amt had been subject to independent divergent changes, probably driven by their own functional adaptation or selection. Indeed, the knockdown or knockout of Rh vs. Amt found in same organisms produced different phenotypes, as seen in green alga (7,8) or slime mold (21,49). It should be noted that the embryonic phenotypes caused by rhr-1 RNAi cannot be compensated by endogenously expressed amt genes.…”

“…In this organism, Rh1 is specifically induced by 3% CO 2 (4), and its knockdown by RNA interference (RNAi) deters the cells from rapid growth at high CO 2 levels (7). Consistent with this substrate specificity, Rh differs from related ammonium transporters (Amt) in evolutionary history and functional diversification (5), and the phenotypes of an AMT mutant strain and an RH RNAi strain differ in C. reinhardtii (8). Parallel to these studies, evidence that Amt is a gas channel for NH 3 has also accumulated (9-13) and been substantiated by the high-resolution structures of two prokaryotic Amt proteins (14)(15)(16).…”

Natural History of Transposition in the Green Alga Chlamydomonas reinhardtii: Use of the AMT4 Locus as an Experimental System

“…Like other AMT genes, AMT4 is highly expressed under nitrogen-limiting conditions (González-Ballester et al 2004) and appears to be the major methylamine transporter. We have shown previously that most spontaneous mutations to methylamine (methylammonium) resistance occur in AMT4 and have briefly described several transposon-induced lesions (Kim et al 2005).…”

“…The sequence of one, which we propose to call the Bill element, indicates that it probably transposes by way of a DNA intermediate and requires functions that it does not encode. The sequence of the other and bioinformatic analysis indicates that it derives from a miniature retrotransposon or TRIM, which we propose to call MRC1 (miniature retrotransposon of Chlamydomonas).T HE AMT4 gene of Chlamydomonas reinhardtii codes for an NH 3 gas channel that also transports the toxic analog methylamine (CH 3 NH 2 ) (Kim et al 2005). Like other AMT genes, AMT4 is highly expressed under nitrogen-limiting conditions (González-Ballester et al 2004) and appears to be the major methylamine transporter.…”

“…Like other AMT genes, AMT4 is highly expressed under nitrogen-limiting conditions (González-Ballester et al 2004) and appears to be the major methylamine transporter. We have shown previously that most spontaneous mutations to methylamine (methylammonium) resistance occur in AMT4 and have briefly described several transposon-induced lesions (Kim et al 2005).Transposable elements can be classified by their replicative intermediates: class I transposons, the retrotransposons, replicate by means of an RNA intermediate, whereas class II transposons replicate through a DNA intermediate (Sabot et al 2004). Members of both these major families have been described in C. reinhardtii.…”