title: 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.
authors.affiliation: VTT Technical Research Centre of Finland, Espoo, Finland.
authors.forename: John
authors.initials: J
authors.lastname: Londesborough
authors.forename: Peter
authors.initials: P
authors.lastname: Richard
authors.forename: Mari
authors.initials: M
authors.lastname: Valkonen
authors.forename: Kaarina
authors.initials: K
authors.lastname: Viljanen
mesh_terms: D020816:Amino Acid Motifs; D001089:Arabinose / Q000378:metabolism*; D001692:Biological Transport; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering; D010429:Pentoses / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D004718:Saccharomycetales / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D010429:Pentoses; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001089:Arabinose