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ScOPT1 and AtOPT4 function as proton-coupled oligopeptide transporters with broad but distinct substrate specificities.

ABSTRACT: A group of OPTs (oligopeptide transporters) exclusively identified in plants and fungi are proposed to transport oligopeptides and derivatives of three to six amino acids in length, but their transport mechanisms and biological functions are poorly understood. We expressed the Saccharomyces cerevisiae (yeast) OPT ScOPT1 and five Arabidopsis thaliana AtOPTs in Xenopus laevis oocytes for two-electrode voltage-clamp studies. ScOPT1 produced inward currents in response to GSH or GSSG, the phytochelatin (PC) PC2 and oligopeptides including the tetrapeptide GGFL, but not KLGL. Inward currents were dependent on the external proton and substrate concentrations, with high affinity for both. This and the inward currents evoked by substrates with net negative charges showed that ScOPT1 is a proton-coupled transporter. ScOPT1 displayed highest apparent affinity for PC2, with small differences in the maximal current among substrates. Glutathione transport by any of the tested AtOPTs, including AtOPT6, was not detected in yeast growth complementation assays. With AtOPT4, initially only small KLGL-dependent currents were recorded in batches of oocytes showing high ScOPT1 expression. AtOPT4 expression was optimized by swapping the 5'-untranslated region with that of ScOPT1. AtOPT4 displayed a higher affinity for KLGL than ScOPT1 did for any peptide. AtOPT4-mediated KLGL transport was detectable at pH 5.0, but not at pH 6.0 or 7.0. Taken together, our results demonstrate that ScOPT1 and AtOPT4 are proton-coupled OPTs with broad but distinct substrate specificities and affinities.

SUBMITTER: Osawa H

PROVIDER: S-EPMC1383685 | biostudies-other | 2006 Jan

REPOSITORIES: biostudies-other

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Project description:Fungal Oligopeptide Transporters (Fot) Fot1, Fot2 and Fot3 have been found in Saccharomyces cerevisiae wine strains, but not in strains from other environments. In the S. cerevisiae wine strain EC1118, Fot1 and Fot2 are responsible for a broader range of oligopeptide utilization in comparison with strains not containing any Fot. This leads to better fermentation efficiency and an increased production of desirable organoleptic compounds in wine. Despite the benefits associated with Fot activity in S. cerevisiae within the wine environment, little is known about this family of transporters in yeast. The presence of Fot1, Fot2 and Fot3 in S. cerevisiae wine strains is due to horizontal gene transfer from the yeast Torulaspora microellipsoides, which harbors Fot2Tm, FotX and FotY proteins. Sequence analyses revealed that Fot family members have a high sequence identity in these yeast species. In this work, we aimed to further characterize the different Fot family members in terms of subcellular localization, gene expression in enological fermentation and substrate specificity. Using CRISPR/Cas9, we constructed S. cerevisiae wine strains containing each different Fot as the sole oligopeptide transporter to analyze their oligopeptide preferences by phenotype microarrays. The results of oligopeptide consumption show that Fot counterparts have different di-/tripeptide specificities, suggesting that punctual sequence divergence between FOT genes can be crucial for substrate recognition, binding and transport activity. FOT gene expression levels in different S. cerevisiae wine strains during enological fermentation, together with predicted binding motifs for transcriptional regulators in nitrogen metabolism, indicate that these transporters may be under the control of the Nitrogen Catabolite Repression (NCR) system. Finally, we demonstrated that Fot1 is located in the yeast plasma membrane. This work contributes to a better understanding of this family of oligopeptide transporters, which have demonstrated a key role in the utilization of oligopeptides by S. cerevisiae in enological fermentation.

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ScOPT1 and AtOPT4 function as proton-coupled oligopeptide transporters with broad but distinct substrate specificities.

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