Water channels are important for osmotic adjustments of yeast cells at low temperature

Abstract

The importance of aquaporin expression in water permeability in Saccharomyces cerevisiae was assessed by measuring the osmotic water permeability coefficient (P f ) and the activation energies (E a ) from both hypo-and hypertonic experiments performed with whole protoplasts from four strains differing in aquaporin level of expression: parental, double-deleted and overexpressing AQY1 or AQY2. Double-deleted (lower P f ) and AQY1-overexpressing strains (higher P f ) presented linear Arrhenius plots with E a consistent with fluxes mainly through the lipids [16?3 kcal mol "1 (68?2 kJ mol )], respectively. The Arrhenius plots for the parental (swelling experiments) and overexpressing AQY2 strains (swelling and shrinking experiments) were not linear, presenting a break point with a change in slope around 23 6C. The E a values for these strains, calculated for temperatures ranging from 7 to 23 6C, were lower [9?5 kcal mol "1 (39?7 kJ mol The permeabilities for each strain relative to the deletion strain show that an increase in permeability due to the presence of aquaporins was more relevant at low temperatures. Following our results, we propose that water channels play an important role for osmotic adjustment of yeast cells at low temperature.

Fungal aquaporins: cellular functions and ecophysiological perspectives

“…When human red cells were treated with pchloromercuribenzosulfonic acid to block proteinaceous water transport, similar high osmotic permeability (20 μm/s, Macey et al 1972;Macey and Farmer 1970) was observed. By contrast, protoplasts of yeast mutants, harboring no aquaporin (AQP) capable of water transport, revealed P f values of only 2 μm/s at 23°C (Soveral et al 2006). Together, these results point toward the importance of lipid composition for water permeability of biological membranes in addition to temperature (Finkelstein 1976;Krylov et al 2001;Lande et al 1995;Kleinhans 1998;Redwood and Haydon 1969).…”

Water Transport in Intact Yeast Cells as Assessed by Fluorescence Self-Quenching

“…The aquaporin-encoding genes in S. cerevisiae (AQY1 and AQY2) contain inactivating mutations in most laboratory strains, and only AQY1 encodes a functional water channel in most natural isolates and industrial strains (2,3). In strains with altered expression of these genes, a significant role for yeast aquaporins restricted to low temperatures has been found (5).…”

“…Light scattering stopped-flow methodologies have been used to measure osmotic water permeability (P f ) in several systems, including yeast protoplasts and vesicles (4,5,8,10), but they were not successful when they were used with intact yeast cells (data not shown). With protoplasts, equivalent P f values were obtained using two different stopped-flow techniques, light scattering and fluorescence (5).…”

“…With protoplasts, equivalent P f values were obtained using two different stopped-flow techniques, light scattering and fluorescence (5).…”

“…The strains used were 10560-6B (MAT␣ leu2::hisG trp1::hisG his3::hisG ura3-52), 10560-6B/pYX012 (KanMX) (ϭ ANT29) (referred to below as the parental strain), and 10560-6B/pYX012 (KanMX AQY1-1) (ϭ ANT27) (referred to below as the strain overexpressing AQY1) (6 For loading of the fluorophore we used the protocol described previously (5). Briefly, immediately before an osmotic challenge, nonenergized cells were preloaded for 10 min at 30°C with the membrane-permeable nonfluorescent precursor 5-and 6-carboxyfluorescein diacetate (CFDA) (concentration in isosmotic solution, 1 mM) that is cleaved intracellularly by nonspecific esterases and generates the impermeable fluorescent form.…”