A high affinity nitrate transport system from Chlamydomonas requires two gene products

A nitrate-regulated cluster of genes involved in nitrate transport and assimilation has been identified in Chlamydomonas reinhardtii. Mutant strains of the alga, which are defective in some aspect of transport and assimilation have been used to assign functions to these genes. This analysis has suggested that two gene products are necessary to obtain a functional high affinity nitrate system in Chlamydomonas [Quesada et al, (1994) Plant J. 5, 407-419]. In this paper we have tested this hypothesis by injecting Xenopus oocytes with mRNA prepared from these two cDNAs, Nrt2;1 and Nar2, and then assaying the oocytes for nitrate transport activity. Oocytes injected with single types of mRNA did not show any nitrate transport activity. Furthermore, Nar2 mRNA was toxic to oocytes, with nearly 60% of the oocytes dead 3 days after the injection. However, when oocytes were injected with a mixture of two mRNAs prepared from Nrt2;1 and Nar2, a high affinity nitrate transport activity could be measured. However, the K-m for nitrate of this transport system was 28 mu M which is higher than the value of 1.6 mu M which had been obtained by the analysis of mutant phenotypes. The pi-I-dependence of the nitrate-elicited currents was consistent with a proton-cotransport mechanism. These results prove that two gene products are required to produce a functional high affinity nitrate transport system and that this process does not involve transcriptional regulation. (C) 2000 Federation of European Biochemical Societies.