Dataset Information

Translational autoregulation of the S. cerevisiae high-affinity polyamine transporter Hol1.

ABSTRACT: Polyamines, small organic polycations, are essential for cell viability, and their physiological levels are homeostatically maintained by post-transcriptional regulation of key biosynthetic enzymes. In addition to de novo synthesis, cells can also take up polyamines; however, identifying cellular polyamine transporters has been challenging. Here we show that the S. cerevisiae HOL1 mRNA is under translational control by polyamines, and we reveal that the encoded membrane transporter Hol1 is a high-affinity polyamine transporter and is required for yeast growth under limiting polyamine conditions. Moreover, we show that polyamine inhibition of the translation factor eIF5A impairs translation termination at a Pro-Ser-stop motif in a conserved upstream open reading frame on the HOL1 mRNA to repress Hol1 synthesis under conditions of elevated polyamines. Our findings reveal that polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels.

SUBMITTER: Vindu A

PROVIDER: S-EPMC8500938 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Translational autoregulation of the S. cerevisiae high-affinity polyamine transporter Hol1.

Vindu Arya A Shin Byung-Sik BS Choi Kevin K Christenson Eric T ET Ivanov Ivaylo P IP Cao Chune C Banerjee Anirban A Dever Thomas E TE

Molecular cell 20210809 19

Polyamines, small organic polycations, are essential for cell viability, and their physiological levels are homeostatically maintained by post-transcriptional regulation of key biosynthetic enzymes. In addition to de novo synthesis, cells can also take up polyamines; however, identifying cellular polyamine transporters has been challenging. Here we show that the S. cerevisiae HOL1 mRNA is under translational control by polyamines, and we reveal that the encoded membrane transporter Hol1 is a hig ...[more]

PMID: 34375581

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Project description:L-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Especially at low concentrations of L-arabinose, its uptake via the Gal2 galactose transporter is an important rate-controlling step in the complete conversion of these feedstocks by engineered, pentose-metabolizing Saccharomyces cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least three-fold higher in L-arabinose-limited cultures than in glucose-limited and ethanol-limited cultures. Of five genes that showed an over 30-fold higher transcript level in arabinose-grown cultures, only one (Pc20g01790) restored growth on L-arabinose upon expression in an engineered L-arabinose-fermenting S. cerevisiae strain in which GAL2 had been deleted. Sugar-transport assays indicated that Pc20g01790this transporter, designated as PcAraT, encodes functions as a high-affinity (Km = 0.13 mM) L-arabinose-proton symporter that does not transport xylose or glucose. An L-arabinose-metabolizing S. cerevisiae strain co-expressing Pc20g01790PcAraT and GAL2 showed lower residual substrate concentrations in L-arabinose-limited chemostat cultures (4 mg L-1) than a congenic strain in which L-arabinose import exclusively depended on Gal2 (1.8 g L-1). Inhibition of L-arabinose transport by these sugars was less pronounced than observed with Gal2. A hexose-phosphorylation-deficient, L-arabinose-metabolizing S. cerevisiae strain expressing PcAraT Pc20g0190 grew on 20 g L-1 L-arabinose in the presence of 20 g L-1 glucose, which completely inhibited growth on L-arabinose of a congenic strain dependent on L-arabinose transport via Gal2. Its high affinity and specificity for L-L-arabinose, combined with limited sensitivity to inhibition by glucose and D-D-xylose make PcAraT/ Pc20g01790 a valuable transporter gene for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

Project description:Backgroundl-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains.ResultsChemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (Km = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10-3 and 1.8 g L-1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L-1 l-arabinose and 20 g L-1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2.ConclusionIts high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

Dataset Information

Translational autoregulation of the S. cerevisiae high-affinity polyamine transporter Hol1.

Publications

Translational autoregulation of the S. cerevisiae high-affinity polyamine transporter Hol1.

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