Structural features of the uniporter/symporter/antiporter superfamily

Goswitz, Visala Chepuri; Brooker, Robert J.

doi:10.1002/pro.5560040319

Cited by 92 publications

(13 citation statements)

References 21 publications

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“…Solute transporters such as AspT and OxlT have a network of key amino acid residues that facilitate substrate movement into and out of an appropriate binding site, thereby defining a substrate translocation pathway through the protein (48,49). For transporters of polar molecules like aspartate, this pathway is assumed to be enriched in residues of a more hydrophilic character than found elsewhere in the transmembrane helices, as is the case for the substrate translocation pathways found in other transporters (12,20,25,29,49). For these reasons, because TM3 is enriched with hydrophilic amino acid residues, including aspartate 67 and arginine 76, and because these are the only charged residues in the TMs of AspT, TM3 deserves attention.…”

Section: Discussionmentioning

confidence: 99%

“…We caution, however, that these two explanations are not mutually exclusive. A transmembrane helix might have positions that face other helices or the hydrophilic translocation pathway (e.g., see the work of Goswitz and Brooker [20]); these positions might be inaccessible but chemically reactive. In contrast, there might be positions that face the membrane lipid, and these positions might be accessible but chemically unreactive.…”

Section: Vol 191 2009 Cysteine Scanning Of Tm3 Of Aspt 2127mentioning

confidence: 99%

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Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT

2009

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AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of L-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of L-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, L-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes L-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a functional unit.In some strains of the lactic acid bacterium Tetragenococcus halophilus, a proton motive force (PMF) is generated by the combined action of an intracellular L-aspartate decarboxylation reaction catalyzed by an L-aspartate-4-decarboxylase (AspD [EC 4.1. The PMF generated is sufficiently high to drive ATP synthesis via the bacterial F o F 1 ATPase. This combination of PMF and ATP synthesis has been proposed as a proton motive metabolic cycle, and the prototype model is found in Oxalobacter formigenes (3, 9, 30). Such decarboxylation reactions are thought to be advantageous for cells because the reactions generate metabolic energy and regulate the intracellular pH. In previous works using proteoliposomes, we found that the aspartate:alanine exchange catalyzed by AspT is electrogenic (1, 2). The biochemical features of substrate transport by AspT indicate that the protein can be classified as a conventional secondary transport protein and that it is an electrogenic antiporter similar to the prototype precursor:product exchanger OxlT, an oxal...

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Section: Discussionmentioning

confidence: 99%

Section: Vol 191 2009 Cysteine Scanning Of Tm3 Of Aspt 2127mentioning

confidence: 99%

Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT

2009

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“…However, not all transmembrane helices of Pho84 are involved in the lining of the putative translocation channel. For the members belonging to the MFS, helices 1, 2, 4, 5, 7, 8, 10 and 11 are predicted to be channel-lining domains [41]. Since Pho84 is a member of the MFS, we selected only these helices for further analysis.…”

Section: Rationale Of the Mutagenesis Designmentioning

confidence: 99%

Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H+ transceptor and its effect on signalling to the PKA and PHO pathways

Samyn

Ruiz-Pavón

Andersson

et al. 2012

Biochemical Journal

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In Saccharomyces cerevisiae, the Pho84 phosphate transporter acts as the main provider of phosphate to the cell using a proton symport mechanism, but also mediates rapid activation of the PKA (protein kinase A) pathway. These two features led to recognition of Pho84 as a transceptor. Although the physiological role of Pho84 has been studied in depth, the mechanisms underlying the transport and sensor functions are unclear. To obtain more insight into the structure-function relationships of Pho84, we have rationally designed and analysed site-directed mutants. Using a three-dimensional model of Pho84 created on the basis of the GlpT permease, complemented with multiple sequence alignments, we selected Arg(168) and Lys(492), and Asp(178), Asp(358) and Glu(473) as residues potentially involved in phosphate or proton binding respectively, during transport. We found that Asp(358) (helix 7) and Lys(492) (helix 11) are critical for the transport function, and might be part of the putative substrate-binding pocket of Pho84. Moreover, we show that alleles mutated in the putative proton-binding site Asp(358) are still capable of strongly activating PKA pathway targets, despite their severely reduced transport activity. This indicates that signalling does not require transport and suggests that mutagenesis of amino acid residues involved in binding of the co-transported ion may constitute a promising general approach to separate the transport and signalling functions in transceptors.

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“…Distinctly, LplT moves LPL using a passive diffusion mechanism which was not inhibited by arsenate blocking of ATP production or by carbonyl cyanide m-chlorophenylhydrazone, a protonophore that dissipates a proton gradient across the IM (14). MFS is also called the uniporter-symporter-antiporter family (76,80). A uniporter promotes equilibrium of substrate across the membrane simply by following a substrate concentration gradient.…”

Section: Identification Of Lplt In Gram-negative Bacteriamentioning

confidence: 99%

Biogenesis, transport and remodeling of lysophospholipids in Gram-negative bacteria

Zheng

Lin

et al. 2017

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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Lysophospholipids (LPLs) are metabolic intermediates in bacterial phospholipid turnover. Distinct from their diacyl counterparts, these inverted cone-shaped molecules share physical characteristics of detergents, enabling modification of local membrane properties such as curvature. The functions of LPLs as cellular growth factors or potent lipid mediators have been extensively demonstrated in eukaryotic cells but are still undefined in bacteria. In the envelope of Gram-negative bacteria, LPLs are derived from multiple endogenous and exogenous sources. Although several flippases that move non-glycerophospholipids across the bacterial inner membrane were characterized, lysophospholipid transporter LplT appears to be the first example of a bacterial protein capable of facilitating rapid retrograde translocation of lyso forms of glycerophospholipids across the cytoplasmic membrane in Gram-negative bacteria. LplT transports lyso forms of the three bacterial membrane phospholipids with comparable efficiency, but excludes other lysolipid species. Once a LPL is flipped by LplT to the cytoplasmic side of the inner membrane, its diacyl form is effectively regenerated by the action of a peripheral enzyme, acyl-ACP synthetase/LPL acyltransferase (Aas). LplT-Aas also mediates a novel cardiolipin remodeling by converting its two lyso derivatives, diacyl or deacylated cardiolipin, to a triacyl form. This coupled remodeling system provides a unique bacterial membrane phospholipid repair mechanism. Strict selectivity of LplT for lyso lipids allows this system to fulfill efficient lipid repair in an environment containing mostly diacyl phospholipids. A rocker-switch model engaged by a pair of symmetric ion-locks may facilitate alternating substrate access to drive LPL flipping into bacterial cells.

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Structural features of the uniporter/symporter/antiporter superfamily

Cited by 92 publications

References 21 publications

Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT

Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT

Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H+ transceptor and its effect on signalling to the PKA and PHO pathways

Biogenesis, transport and remodeling of lysophospholipids in Gram-negative bacteria

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