The Hrk1 kinase is a determinant of acetic acid tolerance in yeast by modulating H+ and K+ homeostasis.

Acetic acid-induced stress is a common challenge in natural environments and industrial bioprocesses, significantly affecting the growth and metabolic performance of Saccharomyces cerevisiae. The adaptive response and tolerance to this stress involves the activation of a complex network of molecular pathways. This study aims to delve deeper into these mechanisms in S. cerevisiae, particularly focusing on the role of the Hrk1 kinase. Hrk1 is a key determinant of acetic acid tolerance, belonging to the NPR/Hal family, whose members are implicated in the modulation of the activity of plasma membrane transporters that orchestrate nutrient uptake and ion homeostasis. The influence of Hrk1 on S. cerevisiae adaptation to acetic acid-induced stress was explored by employing a physiological approach based on previous phosphoproteomics analyses. The results from this study reflect the multifunctional roles of Hrk1 in maintaining proton and potassium homeostasis during different phases of acetic acid-stressed cultivation. Hrk1 is shown to play a role in the activation of plasma membrane H+-ATPase, maintaining pH homeostasis, and in the modulation of plasma membrane potential under acetic acid stressed cultivation. Potassium (K+) supplementation of the growth medium, particularly when provided at limiting concentrations, led to a notable improvement in acetic acid stress tolerance of the hrk1Δ strain. Moreover, abrogation of this kinase expression is shown to confer a physiological advantage to growth under K+ limitation also in the absence of acetic acid stress. The involvement of the alkali metal cation/H+ exchanger Nha1, another proposed molecular target of Hrk1, in improving yeast growth under K+ limitation or acetic acid stress, is proposed.

The second intracellular loop of the yeast Trk1 potassium transporter is involved in regulation of activity, and interaction with 14-3-3 proteins.

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.

Isolation and Characterization of Chitosans from Different Fungi with Special Emphasis on Zygomycetous Dimorphic Fungus Benjaminiella poitrasii: Evaluation of Its Chitosan Nanoparticles for the Inhibition of Human Pathogenic Fungi.

The cell wall chitosan was extracted from fungi belonging to different taxonomic classes, namely, Benjaminiella poitrasii (Zygomycetes, dimorphic), Hanseniaspora guilliermondii, Issatchenkia orientalis, Pichia membranifaciens, and Saccharomyces cerevisiae (Ascomycetes, yeasts), and Agaricus bisporus and Pleurotus sajor-caju (Basidiomycetes). The maximum yield of chitosan was 60.89 ± 2.30 mg/g of dry mycelial biomass of B. poitrasii. The degree of deacetylation (DDA) of chitosan extracted from different fungi, as observed with 1H NMR, was in the range of 70-93%. B. poitrasii chitosan exhibited the highest DDA (92.78%). The characteristic absorption bands were observed at 3450, 1650, 1420, 1320, and 1035 cm-1 by FTIR. Compared to chitosan from marine sources (molecular weight, MW, 585 kDa), fungal chitosans showed lower MW (6.21-46.33 kDa). Further, to improve the efficacy of B. poitrasii chitosan (Bp), nanoparticles (Np) were synthesized using the ionic gelation method and characterized by dynamic light scattering (DLS). For yeast and hyphal chitosan nanoparticles (BpYCNp and BpHCNp), the average particle size was <200 nm with polydispersity index of 0.341 ± 0.03 and 0.388 ± 0.002, respectively, and the zeta potential values were 21.64 ± 0.34 and 24.48 ± 1.58 mV, respectively. The B. poitrasii chitosans and their nanoparticles were further evaluated for antifungal activity against human pathogenic Candida albicans ATCC 10231, Candida glabrata NCYC 388, Candida tropicalis ATCC 750, Cryptococcus neoformans ATCC 34664, and Aspergillus niger ATCC 10578. BpHCNps showed lower MIC90 values (0.025-0.4 mg/mL) than the chitosan polymer against the tested human pathogens. The study suggested that nanoformulation of fungal chitosan, which has low molecular weight and high % DDA, is desirable for antifungal applications against human pathogens. Moreover, chitosans as well as their nanoparticles were found to be hemocompatible and are therefore safe for healthcare applications.

Dimerisation of the Yeast K+ Translocation Protein Trk1 Depends on the K+ Concentration.

In baker's yeast (Saccharomyces cerevisiae), Trk1, a member of the superfamily of K-transporters (SKT), is the main K+ uptake system under conditions when its concentration in the environment is low. Structurally, Trk1 is made up of four domains, each similar and homologous to a K-channel α subunit. Because most K-channels are proteins containing four channel-building α subunits, Trk1 could be functional as a monomer. However, related SKT proteins TrkH and KtrB were crystallised as dimers, and for Trk1, a tetrameric arrangement has been proposed based on molecular modelling. Here, based on Bimolecular Fluorescence Complementation experiments and single-molecule fluorescence microscopy combined with molecular modelling; we provide evidence that Trk1 can exist in the yeast plasma membrane as a monomer as well as a dimer. The association of monomers to dimers is regulated by the K+ concentration.

MPM motifs of the yeast SKT protein Trk1 can assemble to form a functional K+-translocation system.

The yeast Trk1 polypeptide, like other members of the Superfamily of K Transporters (SKT proteins) consists of four Membrane-Pore-Membrane motifs (MPMs A-D) each of which is homologous to a single K-channel subunit. SKT proteins are thought to have evolved from ancestral K-channels via two gene duplications and thus single MPMs might be able to assemble when located on different polypeptides. To test this hypothesis experimentally we generated a set of partial gene deletions to create alleles encoding one, two, or three MPMs, and analysed the cellular localisation and interactions of these Trk1 fragments using GFP tags and Bimolecular Fluorescence Complementation (BiFC). The function of these partial Trk1 proteins either alone or in combinations was assessed by expressing the encoding genes in a K+-uptake deficient strain lacking also the K-channel Tok1 (trk1,trk2,tok1Δ) and (i) analysing their ability to promote growth in low [K+] media and (ii) by ion flux measurements using "microelectrode based ion flux estimation" (MIFE). We found that proteins containing only one or two MPM motifs can interact with each other and assemble with a polypeptide consisting of the rest of the Trk system to form a functional K+-translocation system.

The S. cerevisiae cation translocation protein Trk1 is functional without its "long hydrophilic loop" but LHL regulates cation translocation activity and selectivity.

In Saccharomyces cerevisiae, K+-uptake under K+-limiting conditions is largely mediated by the cation translocation systems Trk1 and Trk2 belonging to the family of SKT proteins. They are related to two-transmembrane-domain (inward rectifying K-) channels but unlike the symmetrical tetrameric structure of K-channels, a single Trk contains four pore-forming domains (A-D) encoded on one polypeptide chain. Between domains A and B Trks contain large cytosolic regions dubbed "long hydrophilic loop" (LHL). LHLs are not homologous/similar to any other identified protein (domain) and also show little similarity between Trk1 and Trk2. Here we demonstrate that Trk1 is functional without LHL. However, in growth experiments NaCl sensitivity of Trk1[ΔLHL] expressing cells is increased under K+-limiting conditions compared to full-length Trk1. Non-invasive ion flux measurements showed that K+-influx through Trk1 and Trk1[ΔLHL] is decreased in the presence of surplus Na+ due to permeability of the proteins for both cations and competition between them. Trk1[ΔLHL] is less affected than full-length Trk1 because it is more selective for K+ over Na+. Furthermore, K+ re-uptake after starvation is delayed and decreased in Trk1[ΔLHL]. Thus, a role of LHL is regulating cation fluxes through Trk1 by (i) allowing also Na+ to pass if monovalent cations (mainly K+) are limiting and (ii) by accelerating/enhancing a switch from low to high affinity ion translocation. We propose that LHL could modulate Trk1 transport properties via direct influence on a transmembrane helix (M2A) which can switch between bent and straight conformation, thereby directly modifying the radius of the pore and selectivity filter.