Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants

Abstract

Summary Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. In Saccharomyces cerevisiae , the high K + content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters formerly believed to extrude K + from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p and presumed as a putative plasma membrane K + /H + antiporter. We prepared kha1 and tok1-kha1 deletion strains in the B31 and MAB 2d background. Both the strains contain the ena1-4 and nha1 deletions; that means they lack the main active sodium and potassium efflux systems. MAB 2d has additional trk1 and trk2 deletions, i.e. is impaired in active K + uptake as well. We performed a large physiological study with these strains to specify the phenotype of kha1 deletion. In our experiments, no difference in K + content or efflux was observed in strains lacking the KHA1 gene compared with control strains. Two main phenotype manifestations of the kha1 deletion were growth defect on high external pH and hygromycin sensitivity. The correlation between these phenotypes and the kha1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of green fluorescent protein (GFP)-tagged Kha1p showed that this antiporter is localized preferentially intracellularly (in contrast to the plasma membrane Na + /H + antiporter Nha1p). Based on these findings, Kha1p is probably not localized in plasma membrane and does not mediate efflux of alkali metal cations from cells, but is important for the regulation of intracelular cation homeostasis and optimal pH control, similarly as the Nhx1p.

Characterization of the Sodium/Hydrogen Exchanger NHA2

“…[17][18][19] As expected, the host strain AXT3 transformed with the empty vector is unable to grow at 100 mM extracellular Na ϩ , at both extracellular pH of 4.0 and 7.4 ( Figure 7A). Expression of prevacuolar (Nhx1) and plasmalemmal (Nha1) S. cerevisiae NHE and human NHA2 all rescue the salt-sensitive phenotype at pH 4 but not at pH 7.4.…”

Identification and Characterization of Vnx1p, a Novel Type of Vacuolar Monovalent Cation/H+ Antiporter of Saccharomyces cerevisiae

“…A pre-vacuolar-bound Na ϩ /H ϩ antiporter (Nhx1p) mediates cation/H ϩ exchange in prevacuoles and possibly in other intracellular compartments (12,16). More recently, a K ϩ /H ϩ exchanger (Kha1p) at the Golgi was reported, and its role in pH regulation was postulated (10,11). Although in S. cerevisiae, vacuolar Na ϩ (and K ϩ ) transport has been attributed mainly to Nhx1p (14), our data and other reports (18,19) Our results clearly indicated that Vnx1 mediated Na ϩ , K ϩ , and Li ϩ transport into the vacuole and that the lack of Vnx1p increased the salt sensitivity of the cells.…”

“…Two other Nha1p homologues, Kha1p and Nhx1p, have been identified in S. cerevisiae. Kha1p has been described as a putative K ϩ /H ϩ antiporter, and its deletion induced a growth defect at high external pH and hygromycin (10). Kha1p co-localized with Mntp1p, a Golgi-specific marker (11).…”

Participation of Endomembrane Cation/H+ Exchanger AtCHX20 in Osmoregulation of Guard Cells

“…Yeast mutants with disrupted kha1 gene alone exhibited no obvious phenotype (Maresova and Sychrova, 2005), so we expressed CHX20 in a yeast mutant (KTA40-2). This strain lacks functional vacuolar and PM-localized Na…”

“…1 antiporters, PM Na 1 pumps (Dnhx1 Dnha1 and Dena1-4), as well as the putative K 1 /H 1 exchanger (Dkha1; Maresova and Sychrova, 2005). Strain KTA40-2 is highly sensitive to salt and to high K 1 , so the transformant (KTA40-2-CHX20) was tested for its ability to grow on moderate levels of Na 1 and very high K…”