The MAPK Hog1p modulates Fps1p-dependent arsenite uptake and tolerance in yeast.

Arsenic is widely distributed in nature and all organisms possess regulatory mechanisms to evade toxicity and acquire tolerance. Yet, little is known about arsenic sensing and signaling mechanisms or about their impact on tolerance and detoxification systems. Here, we describe a novel role of the S. cerevisiae mitogen-activated protein kinase Hog1p in protecting cells during exposure to arsenite and the related metalloid antimonite. Cells impaired in Hog1p function are metalloid hypersensitive, whereas cells with elevated Hog1p activity display improved tolerance. Hog1p is phosphorylated in response to arsenite and this phosphorylation requires Ssk1p and Pbs2p. Arsenite-activated Hog1p remains primarily cytoplasmic and does not mediate a major transcriptional response. Instead, hog1delta sensitivity is accompanied by elevated cellular arsenic levels and we demonstrate that increased arsenite influx is dependent on the aquaglyceroporin Fps1p. Fps1p is phosphorylated on threonine 231 in vivo and this phosphorylation critically affects Fps1p activity. Moreover, Hog1p is shown to affect Fps1p phosphorylation. Our data are the first to demonstrate Hog1p activation by metalloids and provides a mechanism by which this kinase contributes to tolerance acquisition. Understanding how arsenite/antimonite uptake and toxicity is modulated may prove of value for their use in medical therapy.

Structural model of carnitine palmitoyltransferase I based on the carnitine acetyltransferase crystal.

CPT I (carnitine palmitoyltransferase I) catalyses the conversion of palmitoyl-CoA into palmitoylcarnitine in the presence of L-carnitine, facilitating the entry of fatty acids into mitochondria. We propose a 3-D (three-dimensional) structural model for L-CPT I (liver CPT I), based on the similarity of this enzyme to the recently crystallized mouse carnitine acetyltransferase. The model includes 607 of the 773 amino acids of L-CPT I, and the positions of carnitine, CoA and the palmitoyl group were assigned by superposition and docking analysis. Functional analysis of this 3-D model included the mutagenesis of several amino acids in order to identify putative catalytic residues. Mutants D477A, D567A and E590D showed reduced L-CPT I activity. In addition, individual mutation of amino acids forming the conserved Ser685-Thr686-Ser687 motif abolished enzyme activity in mutants T686A and S687A and altered K(m) and the catalytic efficiency for carnitine in mutant S685A. We conclude that the catalytic residues are His473 and Asp477, while Ser687 probably stabilizes the transition state. Several conserved lysines, i.e. Lys455, Lys505, Lys560 and Lys561, were also mutated. Only mutants K455A and K560A showed decreases in activity of 50%. The model rationalizes the finding of nine natural mutations in patients with hereditary L-CPT I deficiencies.

Definition by functional and structural analysis of two malonyl-CoA sites in carnitine palmitoyltransferase 1A.

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of l-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.

Structural model of a malonyl-CoA-binding site of carnitine octanoyltransferase and carnitine palmitoyltransferase I: mutational analysis of a malonyl-CoA affinity domain.

Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an "in silico" macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gómez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001-45008). The putative malonyl-CoA domain contained His(340), implicated together with His(131) in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubi, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183-186). When we mutated COT His(131) the IC(50) increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala(332), present in the domain 8 amino acids away from His(340), decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His(277), His(483), and Ala(478) were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro(479), which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.

Identification of conserved amino acid residues in rat liver carnitine palmitoyltransferase I critical for malonyl-CoA inhibition. Mutation of methionine 593 abolishes malonyl-CoA inhibition.

Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA. By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr(314), Asn(464), Ala(478), Met(593), and Cys(608), rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)). To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A. In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT. Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met(593) mutants were prepared: M593A and M593E. The catalytic efficiency (V(max)/K(m)) of CPT I in mutants A478G and C608A and all Met(593) mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met(593) had been mutated, the malonyl-CoA sensitivity was nearly abolished. Mutations in Ala(478), Cys(608), and Thr(314) to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity. Ala(478) is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gómez-Puertas, P., Rubi, B., Clotet, J., Ariño, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473-11480). Met(593) may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported.