Effect of C-terminal protein tags on pentitol and L-arabinose transport by Ambrosiozyma monospora Lat1 and Lat2 transporters in Saccharomyces cerevisiae

Abstract

Functional expression in heterologous hosts is often less successful for integral membrane proteins than for soluble proteins. Here, two Ambrosiozyma monospora transporters were successfully expressed in Saccharomyces cerevisiae as tagged proteins. Growth of A. monospora on l-arabinose instead of glucose caused transport activities of l-arabinose, l-arabitol, and ribitol, measured using l-[1-(3)H]arabinose, l-[(14)C]arabitol, and [(14)C]ribitol of demonstrated purity. A. monospora LAT1 and LAT2 genes were cloned earlier by using their ability to improve the growth of genetically engineered Saccharomyces cerevisiae on l-arabinose. However, the l-arabinose and pentitol transport activities of S. cerevisiae carrying LAT1 or LAT2 are only slightly greater than those of control strains. S. cerevisiae carrying the LAT1 or LAT2 gene fused in frame to the genes for green fluorescent protein (GFP) or red fluorescent protein (mCherry) or adenylate kinase (AK) exhibited large (>3-fold for LAT1; >20-fold for LAT2) increases in transport activities. Lat1-mCherry transported l-arabinose with high affinity (Km ≈ 0.03 mM) and l-arabitol and ribitol with very low affinity (Km ≥ 75 mM). The Lat2-GFP, Lat2-mCherry, and Lat2-AK fusion proteins could not transport l-arabinose but were high-affinity pentitol transporters (Kms ≈ 0.2 mM). The l-arabinose and pentitol transport activities of A. monospora could not be completely explained by any combination of the observed properties of tagged Lat1 and Lat2, suggesting either that tagging and expression in a foreign membrane alters the transport kinetics of Lat1 and/or Lat2 or that A. monospora contains at least one more l-arabinose transporter.

FIG 1

HPLC analysis of l-[1-¹⁴C]arabinose (catalog no. ARC 1041 [American Radiolabeled Chemicals]; lot 011110). A solution containing 1 mg · ml⁻¹ each of d-xylose (X), l-xylulose (Xu), l-arabinose (A), l-arabitol (Atol), and xylitol (Xtol) and 1.9 × 10⁶ cpm · ml of l-[1-¹⁴C]arabinose (catalog no. ARC 1041; lot 011110) was prepared in water, and 45 μl was injected into the HPLC system. After 70 min at a flow rate of 0.15 ml · min⁻¹, 440-μl fractions were collected, and their radioactivities were determined. The retention times of the standards are indicated by the arrows.

FIG 2

Biphasic kinetics of l-arabitol transport into A. monospora. The initial transport rates (V) were measured between 0.05 and 50 mM l-arabitol. The Eadie-Hofstee plot shows experimental points (●) fitted by curves calculated for the sum of either two Michaelis-Menten systems, a high-affinity system with a K_m of 0.16 mM and a V_max of 3.9 μmol · min⁻¹ · g (dry weight) yeast⁻¹ plus a low-affinity system with a K_m of 75 mM and a V_max of 49 μmol · min⁻¹ · g (dry weight) yeast⁻¹ (dashed line) or the same high-affinity system plus a first-order system with a k of 0.55 μmol · min⁻¹ · g (dry weight) yeast⁻¹ · mM l-arabitol⁻¹ (dotted line).

FIG 3

Inhibition by l-arabinose of pentitol transport by A. monospora. Pentitol transport rates measured at 10 and 0.2 mM pentitol with or without l-arabinose are expressed as percentages of the rates without l-arabinose. Error bars show the standard deviations (SDs) of independent duplicate or triplicate assays.

FIG 4

Effect of C-terminal tags on the transport activities of Lat1 and Lat2. Initial transport rates were assayed at 10 mM or 0.2 mM pentitol or 5 mM l-arabinose (labeled with repurified l-[1-³H]arabinose). Error bars show the SDs of 2 to 7 independent replicates. Control strains include both untransformed hosts and hosts carrying empty plasmids. Transformants carried plasmids containing the unmodified transporter genes (pLAT1 and pLAT2) or transporter genes fused in frame to sequences encoding red fluorescent protein, green fluorescent protein, or adenylate kinase (pLAT1-mCherry, pLAT2-mCherry, pLAT2-GFP, or pLAT2-AK). A. monospora was grown on 40 g l-arabinose · liter⁻¹, and the other yeasts were grown on 20 g glucose · liter⁻¹.

FIG 5

Pentitol concentration dependency of Lat1 and Lat2 fusion proteins. (Left) Effect of substrate concentration on ribitol transport by strains H4388 and H4390, which bear LAT1-mCherry and LAT2-GFP, respectively, on multicopy plasmids, and on l-arabitol transport by strain H4388. The slopes of the trend lines for ribitol and l-arabitol transport by Lat1-mCherry are 0.11 and 0.23 μmol · min⁻¹ · g (dry weight) yeast⁻¹ · mM⁻¹, respectively. (Right) Eadie-Hofstee plots (0.1 to 10 mM substrate) for ribitol transport by strain H4390 and l-arabitol transport by strain H4394 (integrated LAT2-GFP).