Genetic Approaches to Electrogenic Proton Transport by a Yeast H<sup>+</sup>-ATPase

Ds, Perlin; Seto-Young, Donna; Monk, Brian C.; Sl, Harris; S, Na; Haber, James E.

doi:10.1021/ba-1994-0235.ch014

Cited by 5 publications

(8 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In several of these strains, specifically the hal4 hal5 and trk1 trk2 double mutants, sensitivity to toxic cations like hygromycin B can be suppressed by the addition of K + Madrid et al, 1998;Mulet et al, 1999), an effect also observed in the arl1 mutant. By contrast, mutations in other genes, including PMA1 (Goossens et al, 2000;Perlin et al, 1988;Perlin et al, 1989;Serrano et al, 1986) and the phosphatases PPZ1 and PPZ2 (Yenush et al, 2002), led to toxic cation tolerance and decreased potential across the plasma membrane. Because the hyperpolarization phenotype of the arl1 mutant is reversed by the inclusion of a depolarizing cation, K + , our findings are consistent with a model in which the plasma membrane of the arl1 strain is hyperpolarized as a result of disturbances in K + influx rather than H + efflux.…”

Section: Discussionmentioning

confidence: 99%

YeastARL1encodes a regulator of K+ influx

Munson

Haydon

Love

et al. 2004

Journal of Cell Science

View full text Add to dashboard Cite

A molecular genetic approach was undertaken in Saccharomyces cerevisiae to examine the functions of ARL1, encoding a G protein of the Ras superfamily. We show here that ARL1 is an important component of the control of intracellular K+. The arl1 mutant was sensitive to toxic cations, including hygromycin B and other aminoglycoside antibiotics, tetramethylammonium ions, methylammonium ions and protons. The hygromycin-B-sensitive phenotype was suppressed by the inclusion of K+ and complemented by wild-type ARL1 and an allele of ARL1 predicted to be unbound to nucleotide in vivo. The arl1 mutant strain internalized ∼25% more [14C]-methylammonium ion than did the wild type, consistent with hyperpolarization of the plasma membrane. The arl1 strain took up 30-40% less 86Rb+ than did the wild type, showing an inability to regulate K+ import properly, contributing to membrane hyperpolarity. By contrast, K+ and H+ efflux were undisturbed. The loss of ARL1 had no effect on the steady-state level or the localization of a tagged version of Trk1p. High copy suppressors of the hygromycin-B phenotype included SAP155, encoding a protein that interacts with the cell cycle regulator Sit4p, and HAL4 and HAL5, encoding Ser/Thr kinases that regulate the K+-influx mediators Trk1p and Trk2p. These results are consistent with a model in which ARL1, via regulation of HAL4/HAL5, governs K+ homeostasis in cells.

show abstract

Section: Discussionmentioning

confidence: 99%

YeastARL1encodes a regulator of K+ influx

Munson

Haydon

Love

et al. 2004

Journal of Cell Science

View full text Add to dashboard Cite

show abstract

“…The reconstituted vesicles were recovered by centrifugation at 250,000 ϫ g for 1 h. The pellet was resuspended in 400 l of the dilution buffer. Proton transport measurements were made according to the method of Perlin et al (28). A fluorescence quenching reaction volume consisted of 1 ml of 10 mM Hepes-KOH (pH 7.0), 50 mM KCl, 5 mM ATP, 1 g/ml valinomycin, 1.5 M acridine orange, and 50 g of reconstituted vesicles.…”

Section: Methodsmentioning

confidence: 99%

“…Other Procedures-Protein was determined by a modified Lowry method (28). Yeast transformants were prepared by the lithium acetate treatment method, as described in the alkali-cation kit (Bio 101, Inc.).…”

Section: Methodsmentioning

confidence: 99%

Genetic Probing of the First and Second Transmembrane Helices of the Plasma Membrane H+-ATPase from Saccharomyces cerevisiae

Seto-Young¹,

Hall²,

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

Structural features of the putative helical hairpin region comprising transmembrane segments 1 (TM1) and 2 (TM2) of the yeast plasma membrane H(+)-ATPase were probed by site-directed mutagenesis. The importance of phenylalanine residues Phe-116, Phe-119, Phe-120, Phe-126, Phe-144, Phe-159, and Phe-163 was explored by alanine replacement mutagenesis. It was found that substitutions at all positions, except Phe-120 and Phe-144, produced viable enzymes, although a range of cellular growth phenotypes were observed like hygromycin B resistance and low pH sensitivity, which are linked to in vivo action of the H(+)-ATPase. Lethal positions Phe-120 and Phe-144, could be replaced with tryptophan to produce viable enzyme, although the F144W mutant was highly perturbed. ATP hydrolysis measurements showed that Km was not significantly altered for most mutant enzymes, whereas Vmax was moderately reduced with two mutants, F144W and F163A, showing less than 50% of the normal activity. Double Phe-->Ala mutations in TM1 and TM2 were constructed to examine whether such substitutions would result in a higher degree of enzyme destabilization. Mutant F116A/F119A was viable and gave a normal phenotype, while F159A/F163A was not viable. Other double mutants, F116A/F159A and F119AF/159A, which are predicted to lie juxtaposed on TM1 and TM2, produced non-functional enzymes. However, a viable F119V/F159A mutant was isolated and showed hygromycin B resistance. These results suggest that double mutations eliminating 2 phenylalanine residues strongly destabilize the enzyme. A putative proline kink at Gly-122/Pro-123 in TM1 is not essential for enzyme action since these residues could be variously substituted (G122A or G122N; P123A, P123G, or P123F) producing viable enzymes with moderate effects on in vitro ATP hydrolysis or proton transport. However, several substitutions produced prominent growth phenotypes, suggesting that local perturbations were occurring. The location of Pro-123 is important because Gly-122 and Pro-123 could not be exchanged. In addition, a double Pro-Pro created by a G122P mutation was lethal, suggesting that maintenance of an alpha-helical structure is important. Other mutations in the hairpin, including modification of a buried charged residue, E129A, were not critical for enzyme action. These data are consistent with the view that the helical hairpin comprising TM1 and TM2 has important structural determinants that contribute to its overall stability and flexibility.

show abstract

“…Through mutagenesis experiments, we demonstrated that the ATPase function of this protein is necessary for neomycin resistance. Unlike the plasma membrane H+-ATPase, which had been implicated in the uptake of small positively charged antibiotics (Ulaszewski et al, 1987 ;Perlin et al, 1988Perlin et al, , 1989, or the PDRS-encoded protein, which when overexpressed results in altered transport of several antibiotics (Leppert e t al., 1990 ;Balzi e t al., 1994 ;Bissinger & Kuchler, 1994), the NEO7 ATPase appears to more specifically affect aminoglycoside uptake or function. It is unlikely that Neolp plays a role specific to mitochondrial respiratory function, since the disruption of NE07 was lethal even in fermentable media, in which oxidative phosphorylation and mitochondrial protein synthesis are not essential.…”

Section: Discussionmentioning

confidence: 99%