Pilar Eraso

Activation of yeast plasma membrane ATPase by acid pH during growth.

When yeast grows on media in which a final external pH lower than 4 is attained there is a 2–3‐fold increase in plasma membrane ATPase activity. This acid‐mediated activation produces a 2‐fold increase in the ATPase affinity for ATP but does not modify its optimum pH. The acid‐mediated activation is dependent on the stage of growth, only late logarithmic or stationary cells being capable of being activated by incubation in an acidic buffer. Only cells able to activate the ATPase maintain a constant internal pH when incubated in acidic buffers. It is concluded that acid‐mediated activation of the plasma membrane ATPase is a mechanism of maintaining constant internal pH during growth of yeast on acid media.

Functional Domain Analysis of the Yeast ABC Transporter Ycf1p by Site-directed Mutagenesis

The yeast cadmium factor (Ycf1p) is a vacuolar protein involved in resistance to Cd2+ and to exogenous glutathione S-conjugate precursors in yeast. It belongs to the superfamily of ATP binding cassette transporters, which includes the human cystic fibrosis transmembrane conductance regulator and the multidrug resistance-associated protein. To examine the functional significance of conserved amino acid residues in Ycf1p, we performed an extensive mutational analysis. Twenty-two single amino acid substitutions or deletions were generated by site-directed mutagenesis in the nucleotide binding domains, the proposed regulatory domain, and the fourth cytoplasmic loop. Mutants were analyzed phenotypically by measuring their ability to grow in the presence of Cd2+. Expression and subcellular localization of the mutant proteins were examined by immunodetection in vacuolar membranes. For functional characterization of the Ycf1p variants, the kinetic parameters of glutathione S-conjugated leukotriene C4 transport were measured. Our analysis shows that residues Ile711, Leu712, Phe713, Glu927, and Gly1413 are essential for Ycf1p expression. Five other amino acids, Gly663, Gly756, Asp777, Gly1306, and Gly1311, are critical for Ycf1p function, and two residues, Glu709 and Asp821, are unnecessary for Ycf1p biogenesis and function. We also identify several regulatory domain mutants in which Cd2+ tolerance of the mutant strain and transport activity of the protein are dissociated.

Ycf1-dependent cadmium detoxification by yeast requires phosphorylation of residues Ser908 and Thr911

Yeast cadmium factor (Ycf1), an ATP‐binding cassette (ABC) protein of the multidrug resistance protein subfamily, is a vacuolar GS‐conjugate transporter required for heavy metal and drug detoxification. There is evidence that phosphorylation may play a critical role in the function of ABC transporters from higher organisms. In this work, the possibility of Ycf1 phosphorylation was examined using site‐directed mutagenesis. We demonstrate that Ser908 and Thr911, within the regulatory domain (R domain), are functionally important for Ycf1 transport activity and likely sites for phosphorylation. Mutation of these residues to alanine severely impaired the Ycf1‐dependent cadmium detoxification capacity and transport activity, while replacement by acidic residues (mimicking phosphorylation) significantly suppressed the cadmium resistance and transport defects. Both in vitro treatment of Ycf1 with alkaline phosphatase and changes in the electrophoretic mobility of the S908A, T911A and double mutant S908A/T911A proteins supported the conclusion that Ycf1 is a phosphoprotein. The screening of the yeast kinome identified four protein kinases affecting cadmium detoxification, but none of them was involved directly in the phosphorylation of Ycf1. Our data strongly implicate Ycf1 phosphorylation as a key determinant in cadmium resistance in yeast, a significant finding given that very little is known about phosphorylation of ABC transporters in yeast.

Pitfalls in the measurement of membrane potential in yeast cells using tetraphenylphosphonium

The uptake of the lipophilic cation tetraphenylphosphonium (Ph4P+) by Saccharomyces cerevisiae was measured using yeast grown on glucose and harvested either at the logarithmic or at the stationary phase of growth. When yeast was collected at the stationary phase, Ph4P+ uptake proceeded steadily during several hours until an equilibrium was reached. When yeast was collected in the logarithmic phase of growth, a biphasic uptake was observed. The second phase of uptake began when the glucose of the incubation medium had been exhausted. From experiments in the presence of cycloheximide or chloramphenicol it is concluded that the second phase of Ph4P+ uptake is dependent on the synthesis of some protein(s) repressed by glucose but unrelated with the existence of functional mitochondria. The addition of compounds which collapse the membrane potential provokes an efflux from the yeast cells of the Ph4P+ accumulated both during the first phase and the second phase of uptake. It is concluded that accumulation of Ph4P+ in yeast cells is a complex process and that Ph4P+ cannot be used to give a quantitative measure of the yeast plasma membrane potential.

Screening for mutations in Spanish families with myotonia. Functional analysis of novel mutations in CLCN1 gene

Myotonia congenita is an inherited muscle disorder caused by mutations in the CLCN1 gene, a voltage-gated chloride channel of skeletal muscle. We have studied 48 families with myotonia, 32 out of them carrying mutations in CLCN1 gene and eight carry mutations in SCN4A gene. We have found 26 different mutations in CLCN1 gene, including 13 not reported previously. Among those 26 mutations, c.180+3A>T in intron 1 is present in nearly one half of the Spanish families in this series, the largest one analyzed in Spain so far. Although scarce data have been published on the frequency of mutation c.180+3A>T in other populations, our data suggest that this mutation is more frequent in Spain than in other European populations. In addition, expression in HEK293 cells of the new missense mutants Tyr137Asp, Gly230Val, Gly233Val, Tyr302His, Gly416Glu, Arg421Cys, Asn567Lys and Gln788Pro, demonstrated that these DNA variants are disease-causing mutations that abrogate chloride currents.

Gene expression profiling of yeasts overexpressing wild type or misfolded Pma1 variants reveals activation of the Hog1 MAPK pathway

Dominant negative PMA1 mutants render misfolded proteins that are retained in the endoplasmic reticulum (ER) and slowly degraded by ER‐associated degradation. Accumulation of misfolded proteins in the ER activates an ER‐to‐nucleus signalling pathway termed the unfolded protein response (UPR). We have used a PMA1‐D378T dominant negative mutant to analyse its impact on UPR induction. Our results show that overexpression of the misfolded mutant Pma1 does not lead to activation of the UPR. In addition, in mutants with a constitutively activated UPR the turnover rate of the mutant ATPase is not altered. To determine if the expression of the misfolded mutant protein induces some other kind of response we performed global gene expression analysis experiments in yeasts overexpressing either wild type or dominant lethal PMA1 alleles. The results suggest that the high osmolarity glycerol (Hog1) mitogen‐activated protein kinase (MAPK) pathway is activated by both wild type and mutant ATPases. We show that expression of the PMA1 alleles induces phosphorylation of Hog1 and activation of the Hog1 MAPK cascade. This activation is mediated by the Sln1 branch of the stress‐dependent Hog1 MAPK network. Finally, we show that at least two other plasma membrane proteins are also able to activate the Hog1 MAPK system.

Specific phosphoantibodies reveal two phosphorylation sites in yeast Pma1 in response to glucose

Glucose triggers post-translational modifications of the Saccharomyces cerevisiae plasma membrane H(+)-ATPase (Pma1) that lead to an increase in enzyme activity. The activation results from changes in two kinetic parameters: an increase in the affinity of the enzyme for ATP, depending on Ser899, and an increase in the Vmax involving Ser911/Thr912. Using phosphospecific antibodies, we show that Ser899 and Ser911/Thr912 are phosphorylated in vivo during glucose activation and that protein phosphatase Glc7 is involved in the dephosphorylation of Ser899 upon glucose starvation.

A Dominant Negative Mutant of Pma1 Interferes with the Folding of the Wild Type Enzyme

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER‐associated degradation (ERAD) machinery. Several mutant alleles of PMA1, the gene coding for the plasma membrane H +‐ATPase, render misfolded proteins that are subjected to ERAD. A subset of misfolded PMA1 mutants exhibits a dominant negative effect on yeast growth since, when co‐expressed with the wild type allele, both proteins are retained in the ER and degraded. We have used a PMA1‐D378T dominant lethal allele to analyse the mechanism underlying the retention of the wild type enzyme by the dominant negative mutant. A genetic screen was performed for isolation of intragenic suppressors of PMA1‐D378T allele. This analysis pointed to transmembrane helix 10 (TM10) as an important element in the establishment of the dominant lethality. Deletion of the TM10 was able to suppress not only the PMA1‐D378T but all the dominant lethal alleles tested. Biochemical analyses suggest that dominant lethal proteins obstruct, through TM10, the correct folding of the wild type enzyme leading to its retention and degradation by ERAD.