Characterization of a glycerol/H⁺ symport in the halotolerant yeast Pichia sorbitophila

Abstract

Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25 degrees C, pH 5.0, in glucose-grown cells these were: Km = 0.81 +/- 0.11 mM and Vmax = 634.2 +/- 164.8 mumol h-1 per g (glycerol); Km = 1.28 +/- 0.60 mM and Vmax = 558.6 +/- 100.6 mumol h-1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M-NaCl. An increase in accumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on delta pH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.

Active glycerol uptake is a mechanism underlying halotolerance in yeasts: a study of 42 species

“…Glucose-or glycerolgrown cells were harvested in the mid-exponential growth phase (variable optical density according to the strain) by centrifugation, washed twice and resuspended to a final concentration of " 30 mg ml − " (dry wt) in ice-cold distilled water. The methods used to determine (i) initial rates of proton uptake upon glycerol addition, (ii) initial uptake rates of ["%C]glycerol (Amersham) and (iii) the inhibitory effects of the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone) and ethanol on uptake, have been described before (Lucas et al, 1990 ;Lages & Lucas, 1995 (Amersham) accumulation ratios were determined according to Lages & Lucas (1995).…”

“…Besides the enhancement in production observed under stress, and taking into account that glycerol is a liposoluble molecule that leaks through the yeast plasma membrane, its retention should thus require an active response from the cell. In two different osmotolerant yeast species, Debaryomyces hansenii and Pichia sorbitophila, two different types of constitutive active-transport systems for glycerol have been described and characterized : a Na + \glycerol co-transport (Lucas et al, 1990) and a H + \glycerol symport (Lages & Lucas, 1995). Also in Zygosaccharomyces rouxii, an active-transport system for glycerol has been described .…”

New insights on glycerol transport in Saccharomyces cerevisiae

“…Initial rates of glycerol uptake, external alkalinization upon glycerol addition, as well as in/out accumulation ratios, were determined as previously described [5,9,12,13]. The intracellular volume values used to determine intracellular glycerol molarity have been determined by Lages and Lucas [5].…”

Characterization of glycerol uptake in bloodstream and procyclic forms of Trypanosoma brucei

“…During the last few years several studies dealing with glycerol transport mediated by membrane proteins have been published and it has been shown that glycerol can be taken up by the various transport systems present in eukaryotic cells, including facilitated diffusion [7], proton symport [8] and sodium symport [9]. Protein-facilitated glycerol uptake has been described for higher eukaryotic cells such as rat hepatocytes [10] and for microorganisms such as Neurospora crassa [11] and Streptomyces clavuligerus [12].…”