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Evolutionary mix-and-match with MFS transporters.

ABSTRACT: Major facilitator superfamily (MFS) transport proteins are ubiquitous in the membranes of all living cells, and ?25% of prokaryotic membrane transport proteins belong to this superfamily. The MFS represents the largest and most diverse group of transporters and includes members that are clinically important. A wide range of substrates is transported in many instances actively by transduction of the energy stored in an H(+) electrochemical gradient into a concentration gradient of substrate. MFS transporters are characterized by a deep central hydrophilic cavity surrounded by 12 mostly irregular transmembrane helices. An alternating inverted triple-helix structural symmetry within the N- and C-terminal six-helix bundles suggests that the proteins arose by intragenic multiplication. However, despite similar features, MFS transporters share only weak sequence homology. Here, we show that rearrangement of the structural symmetry motifs in the Escherichia coli fucose permease (FucP) results in remarkable homology to lactose permease (LacY). The finding is supported by comparing the location of 34 point mutations in FucP to the location of mutants in LacY. Furthermore, in contrast to the conventional, linear sequence alignment, homologies between sugar- and H(+)-binding sites in the two proteins are observed. Thus, LacY and FucP likely evolved from primordial helix-triplets that formed functional transporters; however, the functional segments assembled in a different consecutive order. The idea suggests a simple, parsimonious chain of events that may have led to the enormous sequence diversity within the MFS.

SUBMITTER: Madej MG

PROVIDER: S-EPMC3625355 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Evolutionary mix-and-match with MFS transporters.

Madej M Gregor MG Dang Shangyu S Yan Nieng N Kaback H Ronald HR

Proceedings of the National Academy of Sciences of the United States of America 20130325 15

Major facilitator superfamily (MFS) transport proteins are ubiquitous in the membranes of all living cells, and ∼25% of prokaryotic membrane transport proteins belong to this superfamily. The MFS represents the largest and most diverse group of transporters and includes members that are clinically important. A wide range of substrates is transported in many instances actively by transduction of the energy stored in an H(+) electrochemical gradient into a concentration gradient of substrate. MFS ...[more]

PMID: 23530251

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Project description:In yeasts, proteins of the Major Superfamily Transporter selectively bind and allow the uptake of sugars to permit growth on varied substrates. The genome of brewer's yeast, Saccharomyces cerevisiae, encodes multiple hexose transporters (Hxt) to transport glucose and other MFS proteins for maltose, galactose, and other monomers. For sugar uptake, the dairy yeast, Kluyveromyces lactis, uses Rag1p for glucose, Hgt1 for glucose and galactose, and Lac12 for lactose. In the related industrial species Kluyveromyces marxianus, there are four genes encoding Lac12-like proteins but only one of them, Lac12, can transport lactose. In this study, which initiated with efforts to investigate possible functions encoded by the additional LAC12 genes in K. marxianus, a genome-wide survey of putative MFS sugar transporters was performed. Unexpectedly, it was found that the KHT and the HGT genes are present as tandem arrays of five to six copies, with the precise number varying between isolates. Heterologous expression of individual genes in S. cerevisiae and mutagenesis of single and multiple genes in K. marxianus was performed to establish possible substrates for these transporters. The focus was on the sugar galactose since it was already reported in K. lactis that this hexose was a substrate for both Lac12 and Hgt1. It emerged that three of the four copies of Lac12, four Hgt-like proteins and one Kht-like protein have some capacity to transport galactose when expressed in S. cerevisiae and inactivation of all eight genes was required to completely abolish galactose uptake in K. marxianus. Analysis of the amino acid sequence of all known yeast galactose transporters failed to identify common residues that explain the selectivity for galactose. Instead, the capacity to transport galactose has arisen three different times in K. marxianus via polymorphisms in proteins that are probably ancestral glucose transporters. Although, this is analogous to S. cerevisiae, in which Gal2 is related to glucose transporters, there are not conserved amino acid changes, either with Gal2, or among the K. marxianus galactose transporters. The data highlight how gene duplication and functional diversification has provided K. marxianus with versatile capacity to utilise sugars for growth.

Project description:Bacterial RNA polymerase is composed of a core of subunits (beta, beta', alpha1, alpha2, omega), which have RNA synthesizing activity, and a specificity factor (sigma), which identifies the start of transcription by recognizing and binding to sequences elements within promoter DNA. Four core promoter consensus sequences, the -10 element, the extended -10 (TGn) element, the -35 element, and the UP elements, have been known for many years; the importance of a nontemplate G at position -5 has been recognized more recently. However, the functions of these elements are not the same. The AT-rich UP elements, the -35 elements ((-35)TTGACA(-30)), and the extended -10 ((-15)TGn(-13)) are recognized as double stranded binding elements, whereas the -5 nontemplate G is recognized in the context of single-stranded DNA at the transcription bubble. Furthermore, the -10 element ((-12)TATAAT(-7)) is recognized as both double strand DNA for the T:A bp at position -12 and as nontemplate, single-strand DNA from positions -11 to -7. The single-strand sequences at positions -11 to -7 as well as the -5 contribute to later steps in transcription initiation that involve isomerization of polymerase and separation of the promoter DNA around the transcription start site. Recent work has demonstrated that the double strand elements may be used in various combinations to yield an effective promoter. Thus, while some minimal number of contacts is required for promoter function, polymerase allows the elements to be mixed and matched. Interestingly, which particular elements are used does not appear to fundamentally alter the transcription bubble generated in the stable complex. In this review, we discuss the multiple steps involved in forming a transcriptionally competent polymerase/promoter complex, and we examine what is known about polymerase recognition of core promoter elements. We suggest that considering promoter elements according to their involvement in early (polymerase binding) or later (polymerase isomerization) steps in transcription initiation rather than simply from their match to conventional promoter consensus sequences is a more instructive form of promoter classification.

Dataset Information

Evolutionary mix-and-match with MFS transporters.

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Evolutionary mix-and-match with MFS transporters.

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