A high-affinity molybdate transporter in eukaryotes

Abstract

Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-bindingcassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K m of Ϸ6 nM, which is the range of the lowest Km values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells. micronutrient transport ͉ molybdenum ͉ molybdenum cofactor ͉ nitrate assimilation M olybdenum is one of the least abundant elements in organisms and is essential for molybdenum cofactor (Moco) biosynthesis. Moco is present in almost all living beings, taking part, as a prosthetic group, in the active site of key enzymes such as nitrate reductase, aldehyde oxidase, xanthine dehydrogenase, and sulfite oxidase. These enzymes participate in crucial processes for life such as nitrate assimilation, phytohormone biosynthesis, purine metabolism, and sulfite detoxification in plants, animals, and microorganisms (1, 2).Moco biosynthesis is a conserved pathway that consists of four main steps (3), conversion of GTP into cyclic pyranopterin monophosphate, synthesis of the molybdopterin dithiolate, adenylation of molybdopterin, and molybdenum-insertion reaction. In the fourth and final step, mature Moco is formed by the ligation of a single Mo atom to adenylated molybdopterin; this process directly depends on the intracellular availability of molybdenum. Deficiency in Moco biosynthesis results in a pleiotropic loss of all Mo-enzymes activity, which leads in humans to a severe metabolic disorder, and affected patients die in early childhood (4).In prokaryotes, molybdenum is taken up in an energydependent process by high-affinity molybdate transporters belonging to the ABC family (5). This system is formed by a periplasmic molybdate-bindin...

Algae and humans share a molybdate transporter

“…Mutants at one of these loci, Nit5, show a reduced molybdate uptake activity together with a tolerance for high concentrations of molybdate or tungstate in presence of any nitrogen source. This phenotype is shown by strain 21gr that has MOT1 as the only molybdate transport activity (11). Mutants at both loci are unable to synthesize Moco and thus are defective in the activity of the molybdoenzyme nitrate reductase (15).…”

“…Proteins related to the bacterial molybdate transport system are not encoded by eukaryotic genomes. Algae (Chlamydomonas) and plants (Arabidopsis) are the only eukaryotes where a MOT1 (molybdate transporter, type 1) protein was demonstrated to transport molybdate with high affinity (11,12). MOT1 mediates a molybdate transport process that exhibits a Km of 7-20 nM and in Chlamydomonas is upregulated by nitrate but is insensitive to external Mo availability.…”

“…MOT1 type proteins are present in bacteria, algae, fungi, and plants but not in animal genomes (11), therefore how animal cells satisfy metabolic molybdenum requirements remains unknown. In animals Moco deficiency is especially serious because it causes a severe phenotype characterized by a progressive neurological damage that leads to death shortly after birth (9,13).…”

“…Mutants at both loci are unable to synthesize Moco and thus are defective in the activity of the molybdoenzyme nitrate reductase (15). In addition, when Chlamydomonas gene MoT1 is knockdown in a wild-type strain, the presence of a putative second transporter balances the uptake of molybdate (11). However, only one member of the MOT1 family is present in the alga genome (11).…”

“…In addition, when Chlamydomonas gene MoT1 is knockdown in a wild-type strain, the presence of a putative second transporter balances the uptake of molybdate (11). However, only one member of the MOT1 family is present in the alga genome (11).…”