Trinucleotide Insertions, Deletions, and Point Mutations in Glucose Transporters Confer K⁺Uptake inSaccharomyces cerevisiae

Abstract

Deletion of TRK1 and TRK2 abolishes high-affinity K ؉ uptake in Saccharomyces cerevisiae, resulting in the inability to grow on typical synthetic growth medium unless it is supplemented with very high concentrations of potassium. Selection for spontaneous suppressors that restored growth of trk1⌬ trk2⌬ cells on K ؉ -limiting medium led to the isolation of cells with unusual gain-of-function mutations in the glucose transporter genes HXT1 and HXT3 and the glucose/galactose transporter gene GAL2.86 Rb uptake assays demonstrated that the suppressor mutations conferred increased uptake of the ion. In addition to K ؉ , the mutant hexose transporters also conferred permeation of other cations, including Na ؉ . Because the selection strategy required such gain of function, mutations that disrupted transporter maturation or localization to the plasma membrane were avoided. Thus, the importance of specific sites in glucose transport could be independently assessed by testing for the ability of the mutant transporter to restore glucose-dependent growth to cells containing null alleles of all of the known functional glucose transporter genes. Twelve sites, most of which are conserved among eukaryotic hexose transporters, were revealed to be essential for glucose transport. Four of these have previously been shown to be essential for glucose transport by animal or plant transporters. Eight represented sites not previously known to be crucial for glucose uptake. Each suppressor mutant harbored a single mutation that altered an amino acid(s) within or immediately adjacent to a putative transmembrane domain of the transporter. Seven of 38 independent suppressor mutations consisted of in-frame insertions or deletions. The nature of the insertions and deletions revealed a striking DNA template dependency: each insertion generated a trinucleotide repeat, and each deletion involved the removal of a repeated nucleotide sequence.

Saccharomyces cerevisiae Multidrug Resistance Transporter Qdr2 Is Implicated in Potassium Uptake, Providing a Physiological Advantage to Quinidine-Stressed Cells

“…The results also show that even after deletion of TRK1, TRK2, and QDR2, there is still K ϩ transport, indicating that other transporters may be involved in K ϩ uptake. It is usually accepted that several nonspecific transporters may be involved in the low-affinity transport of K ϩ observed in the ⌬trk1 ⌬trk2 mutant (22). The existence of a yeast nonselective cation channel responsible for the low-affinity K ϩ uptake was postulated before, although the identity of the protein(s) responsible for this inwardly rectifying pathway is not known (2,3).…”

“…The existence of a yeast nonselective cation channel responsible for the low-affinity K ϩ uptake was postulated before, although the identity of the protein(s) responsible for this inwardly rectifying pathway is not known (2,3). Amino acid or sugar transporters could be involved in this process (22,50) together with Qdr2p and, eventually, other plasma membrane multidrug resistance transporters. Since quinidine strongly decreases the uptake of K ϩ , presumably by acting as an uncoupler at the plasma membrane, the involvement of Qdr2p in the uptake of this essential ion confers a physiological advantage to quinidine-stressed cells.…”

“…However, the nature of this transport remains to be elucidated, although the nonspecific uptake of potassium occurring through sugar and amino acid permeases has been postulated (22,23,50). Based on a number of evidences, Wright et al (50) have considered that amino acid:H ϩ symporters may not have an absolute requirement for H ϩ as a cosubstrate and may be capable of coupling K ϩ ions to drive amino acid uptake.…”

New Plasmid System To Select for Saccharomyces cerevisiae Purine-Cytosine Permease Affinity Mutants

“…It would be of great interest to study these mutations, as they reflect a significant adaptive response to major substrate variations. For instance, some of these mutants may result from the functional modification of other transporters, as has been observed in similar studies: during selection for suppressors that restored potassium uptake in ⌬trk1 ⌬trk2 cells, unusual gain-of-function mutations were characterized in various transporters, such as amino acid permeases Bap2 and Hip1, glucose transporters Hxt1 and Hxt3, and glucose-galactose transporter Gal2 (10,14).…”

A Novel Mechanism of Ion Homeostasis and Salt Tolerance in Yeast: the Hal4 and Hal5 Protein Kinases Modulate the Trk1-Trk2 Potassium Transporter

“…Instead, "low-affinity" uptake pathways allow potassium and other cations to enter the cell under the influence of a negative-inside membrane potential. This uptake may be due to additive contributions of transporters selective for glucose (33,35,39) and other carbon sources, amino acids (76), choline, ammonium, and divalent cations (39), each of which is also likely to transport small cations nonspecifically. Where this has been measured, these transporters exhibit a nonspecific (and marginal) cation transport activity that can be increased by either overexpression or specific mutations within the transmembrane helices (35,39).…”