Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter

Abstract

Candida intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose-proton symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose-H+ symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide-H+ symporters encompassing proteins from several yeasts and filamentous fungi.

Figure 1. Northern blot analysis of GXF1 expression in C. intermedia PYCC 4715 cells cultivated in different carbon sources

The carbon sources (w/v) were: 0.5% xylose (X), 2% glucose (G), 4% xylose (4X), 0.5% lactose (L), 0.5% sorbose (S), 0.5% galactose (GA), 0.5% cellobiose (C), 0.5% (w/v) arabinose (A), or 0.5% trehalose (T). Northern blots were probed with a GXF1-gene-specific probe and, subsequently, with an actin-gene-specific probe, as a loading control.

Figure 2. Glucose and xylose uptake in strain MJY1 (S. cerevisiae TMB 3201 transformed with pGXF1)

Eadie–Hofstee plots of initial uptake rates of (●) D-[¹⁴C]xylose and (▲) D-[¹⁴C]glucose by strain MJY1 cultivated on glucose. dw, dry weight.

Figure 3. Tricine SDS/PAGE (10% polyacrylamide gel, with 0.3% bisacrylamide) of plasma- and mitochondrial-membrane proteins isolated from C. intermedia PYCC 4715 cells cultivated in Verduyn medium containing different concentrations of xylose or glucose as the sole carbon and energy source

The carbon sources were: 0.5% (w/v) xylose (X), 2% (w/v) glucose (G) or 4% (w/v) xylose (4X). The gel was stained with Coomassie Blue. M, molecular mass marker (Sigma–Aldrich, Wide Range); p, plasma membranes; m, mitochondrial membranes.

Figure 4. Amino acid sequence of the N-terminal region of Gxs1p and the degenerate primers used for the 3′ RACE

Figure 5. Northern blot analysis of GXS1 expression in C. intermedia PYCC 4715 cells cultivated in different carbon sources

The carbon sources (w/v) were 0.5% xylose (X), 2% glucose (G) or 4% xylose (4X). Northern blots were probed with a GXS1-gene-specific probe and, subsequently, with an actin-gene-specific probe, as a loading control.

Figure 6. Increases in pH elicited by the addition of xylose (upper panel) or glucose (lower panel) to aqueous cell suspensions of S. cerevisiae strains MJY1 and MJY2 cultivated on glucose

Both sugars were added to a final concentration of 6.7 mM. The arrows indicate the times of sugar addition. X, xylose; G, glucose.

Figure 7. Glucose and xylose uptake mediated by Gxs1p and Gxf1p

Eadie–Hofstee plots of initial uptake rates of (A) D-[¹⁴C]xylose in strain MJY2, and (B) D-[¹⁴C]glucose in strains MJY5 and MJY2 (inset). dw, dry weight.