Growth at High pH and Sodium and Potassium Tolerance in Media above the Cytoplasmic pH Depend on ENA ATPases in Ustilago maydis

Abstract

Potassium and Na؉ effluxes across the plasma membrane are crucial processes for the ionic homeostasis of cells. In fungal cells, these effluxes are mediated by cation/H ؉ antiporters and ENA ATPases. We have cloned and studied the functions of the two ENA ATPases of Ustilago maydis, U. maydis Ena1 (UmEna1) and UmEna2. UmEna1 is a typical K ؉ or Na ؉ efflux ATPase whose function is indispensable for growth at pH 9.0 and for even modest Na ؉ or K ؉ tolerances above pH 8.0. UmEna1 locates to the plasma membrane and has the characteristics of the low-Na ؉ /K ؉ -discrimination ENA ATPases. However, it still protects U. maydis cells in high-Na ؉ media because Na ؉ showed a low cytoplasmic toxicity. The UmEna2 ATPase is phylogenetically distant from UmEna1 and is located mainly at the endoplasmic reticulum. The function of UmEna2 is not clear, but we found that it shares several similarities with Neurospora crassa ENA2, which suggests that endomembrane ENA ATPases may exist in many fungi. The expression of ena1 and ena2 transcripts in U. maydis was enhanced at high pH and at high K ؉ and Na ؉ concentrations. We discuss that there are two modes of Na ؉ tolerance in fungi: the high-Na ؉ -content mode, involving ENA ATPases with low Na ؉ /K ؉ discrimination, as described here for U. maydis, and the low-Na ؉ -content mode, involving Na ؉ -specific ENA ATPases, as in Neurospora crassa.Potassium is the most abundant cation in all types of living cells. Na ϩ , which is fairly abundant in many natural environments, can partially substitute for K ϩ but becomes toxic above a certain Na ϩ /K ϩ ratio (47). Therefore, the homeostatic processes that regulate the steady-state concentrations of K ϩ and Na ϩ in cells as well as the systems that mediate the transport of these cations across the plasma membrane and some endomembranes are crucial for maintaining cell viability. Among all the transport processes involved, K ϩ and Na ϩ effluxes play an indispensable role, and therefore, they take place in all types of living cells. For instance, in animal cells, an essential Na,KATPase that mediates Na ϩ efflux and K ϩ uptake consumes 20 to 30% of the produced ATP (33). Fungal and plant cells do not have this animal-type Na,K-ATPase (10), but K ϩ or Na ϩ efflux ATPases, also called ENA ATPases, are present in every fungal species (12) and have also been described for some bryophytes (15). ENA ATPases are phylogenetically close to but functionally different from animal Na,K-ATPases. Unlike the latter, ENA ATPases pump out almost every alkali cation and not exclusively Na ϩ , and they do not mediate K ϩ uptake (14). The cation promiscuity of ENA ATPases may be an advantage in fungi because their membrane potential is very negative, and they can live in environments with high K ϩ concentrations, such as plant tissues or plant debris. In these environments, the energetic conditions that prevail for K ϩ efflux are similar to those prevailing for Na ϩ efflux in Na ϩ environments (12). Fungal ENA ATPases are, in most cases, not essential in acidic...

Role of ENA ATPase in Na+ efflux at high pH in bryophytes

“…Drops of cell suspensions of three serial ten-fold dilutions were inoculated in the recorded media UmEna2 locates to the ER and is not involved in cellular Na ? efflux (Benito et al 2009). Unfortunately, we have not yet been able to express the PpENA2-GFP and PpENA3-GFP fusions in P. patens protoplasts, and we therefore do not know in which membranes PpENA2 and PpENA3 are expressed.…”

“…or Na ? against their concentration gradients (Benito et al 2009;Idnurm et al 2009). At high pH, the DpH across the plasma membrane would drive H ?…”

Functional analysis of the pH responsive pathway Pal/Rim in the phytopathogenic basidiomycete Ustilago maydis

“…Using fungal Ena-like proteins as queries in the U. maydis database, we identified gene um00204 that showed the highest homology. This gene encodes a protein that shares all the structural characteristics exhibited by Ena proteins, and accordingly was isolated [during the course of our study the same gene was identified and denominated as UmEna2 (Benito et al, 2009)]. Gene expression was evaluated by Northern hybridization in media supplemented with NaCl (0.4 M) or LiCl (0.003 M).…”

Alkali Metal Cation Transport and Homeostasis in Yeasts

“…A recent report (18) has proposed the existence of two types of fungi in regard to Na ϩ tolerance: those tolerant to high Na ϩ contents in the cytoplasm, which include U. maydis, and those intolerant to high Na ϩ contents, which include the model yeast S. cerevisiae. However, in both types, vacuolar transport may play an important role in cation homeostasis.…”

“…The Ena transporters, the main determinants of halotolerance in S. cerevisiae and probably present in most fungi (15,18,19), do not exist in flowering plants (85). Remarkably, two Na ϩ -ATPases in the moss Physcomitrella patens have been characterized (23) and at least one of them (PpEna1), appears to be functional as a sodium pump both in S. cerevisiae (23) and in planta (145).…”