The non-conventional yeast Kluyveromyces marxianus has recently emerged as a promising candidate for many food, environment, and biotechnology applications. This yeast is thermotolerant and has robust growth under many adverse conditions. Here, we show that its ability to grow under potassium-limiting conditions is much better than that of Saccharomyces cerevisiae, suggesting a very efficient and high-affinity potassium uptake system(s) in this species. The K. marxianus genome contains two genes for putative potassium transporters: KmHAK1 and KmTRK1. To characterize the products of the two genes, we constructed single and double knock-out mutants in K. marxianus and also expressed both genes in an S. cerevisiae mutant, that lacks potassium importers. Our results in K. marxianus and S. cerevisiae revealed that both genes encode efficient high-affinity potassium transporters, contributing to potassium homeostasis and maintaining plasma-membrane potential and cytosolic pH. In K. marxianus, the presence of HAK1 supports growth at low K+ much better than that of TRK1, probably because the substrate affinity of KmHak1 is about 10-fold higher than that of KmTrk1, and its expression is induced ~80-fold upon potassium starvation. KmHak1 is crucial for salt stress survival in both K. marxianus and S. cerevisiae. In co-expression experiments with ScTrk1 and ScTrk2, its robustness contributes to an increased tolerance of S. cerevisiae cells to sodium and lithium salt stress.

• Klíčová slova
• Kluyveromyces marxianu, K+–H+ symporter, affinity, potassium, transporter, uniporter,
• MeSH
• draslík * metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• genový knockout MeSH
• Kluyveromyces * genetika   metabolismus   růst a vývoj   MeSH
• koncentrace vodíkových iontů MeSH
• membránové potenciály MeSH
• proteiny přenášející kationty * genetika   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslík * MeSH
• fungální proteiny MeSH
• proteiny přenášející kationty * MeSH

Potassium is an essential intracellular ion, and a sufficient intracellular concentration of it is crucial for many processes; therefore it is fundamental for cells to precisely regulate K+ uptake and efflux through the plasma membrane. The uniporter Trk1 is a key player in K+ acquisition in yeasts. The TRK1 gene is expressed at a low and stable level; thus the activity of the transporter needs to be regulated at a posttranslational level. S. cerevisiae Trk1 changes its activity and affinity for potassium ion quickly and according to both internal and external concentrations of K+, as well as the membrane potential. The molecular basis of these changes has not been elucidated, though phosphorylation is thought to play an important role. In this study, we examined the role of the second, short, and highly conserved intracellular hydrophilic loop of Trk1 (IL2), and identified two phosphorylable residues (Ser882 and Thr900) as very important for 1) the structure of the loop and consequently for the targeting of Trk1 to the plasma membrane, and 2) the upregulation of the transporter's activity reaching maximal affinity under low external K+ conditions. Moreover, we identified three residues (Thr155, Ser414, and Thr900) within the Trk1 protein as strong candidates for interaction with 14-3-3 regulatory proteins, and showed, in an in vitro experiment, that phosphorylated Thr900 of the IL2 indeed binds to both isoforms of yeast 14-3-3 proteins, Bmh1 and Bmh2.

• Klíčová slova
• 14–3–3 proteins, Phosphorylation, Potassium ion uptake, Saccharomyces cerevisiae, Trk1,
• Publikační typ
• časopisecké články MeSH

Yeasts need a high intracellular concentration of potassium to grow. The main K+ uptake system in Saccharomyces cerevisiae is the Trk1 transporter, a complex protein with four MPM helical membrane motifs. Trk1 has been shown to exist in low- or high-affinity modes, which reflect the availability of potassium in the environment. However, when and how the affinity changes, and whether the potassium availability is the only signal for the affinity switch, remains unknown. Here, we characterize the Trk1 kinetic parameters under various conditions and find that Trk1's KT and Vmax change gradually. This gliding adjustment is rapid and precisely reflects the changes in the intracellular potassium content and membrane potential. A detailed characterization of the specific mutations in the P-helices of the MPM segments reveals that the presence of proline in the P-helix of the second and third MPM domain (F820P and L949P) does not affect the function of Trk1 in general, but rather specifically prevents the transporter's transition to a high-affinity state. The analogous mutations in the two remaining MPM domains (L81P and L1115P) result in a mislocalized and inactive protein, highlighting the importance of the first and fourth P-helices in proper Trk1 folding and activity at the plasma membrane.

• Klíčová slova
• Saccharomyces cerevisiae, cation homeostasis, membrane potential, potassium uptake,
• Publikační typ
• časopisecké články MeSH