Stimulation of zero-trans rates of lactose and maltose uptake into yeasts by preincubation with hexose to increase the adenylate energy charge

Pedro M. R. Guimarães (Corresponding Author), Jyri-Pekka Multanen, Lucilia Domingues, Jose A. Teixeira, John Londesborough

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Abstract

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three- to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular β-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [14C]lactose uptake reactions were started. A short (1- to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5- to 5-fold increases in the specific rates of lactose uptake. These increases correlated with increases in EC (from 0.6 to 0.9) and ATP (from 4 to 8 μmol·g dry yeast−1). Stimulation by glucose affected the transport Vmax values, with smaller increases in Km values. Similar observations were made for maltose transport, using a brewer's yeast. These findings suggest that the electrochemical proton potential that drives transport through sugar/H+ symports is significantly lower in the starved yeast suspensions used for zero-trans assays than in actively metabolizing cells. Zero-trans assays with such starved yeast preparations can produce results that seriously underestimate the capacity of sugar/H+ symports. A short exposure to glucose allows a closer approach to the sugar/H+ symport capacity of actively metabolizing cells.
Original languageEnglish
Pages (from-to)3076-3084
JournalApplied and Environmental Microbiology
Volume74
Issue number10
DOIs
Publication statusPublished - 2008
MoE publication typeA1 Journal article-refereed

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