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Molecular determinants of substrate selectivity of a novel organic cation transporter (PMAT) in the SLC29 family.

ABSTRACT: Plasma membrane monoamine transporter (PMAT or ENT4) is a newly cloned transporter assigned to the equilibrative nucleoside transporter (ENT) family (SLC29). Unlike ENT1-3, PMAT mainly functions as a polyspecific organic cation transporter. In this study, we investigated the molecular mechanisms underlying the unique substrate selectivity of PMAT. By constructing chimeras between human PMAT and ENT1, we showed that a chimera consisting of transmembrane domains (TM) 1-6 of PMAT and TM7-11 of hENT1 behaved like PMAT, transporting 1-methyl-4-phenylpyridinium (MPP+, an organic cation) but not uridine (a nucleoside), suggesting that TM1-6 contains critical domains responsible for substrate recognition. To identify residues important for the cation selectivity of PMAT, 10 negatively charged residues were chosen and substituted with alanine. Five of the alanine mutants retained PMAT activity, and four were non-functional due to impaired targeting to the plasma membrane. However, alanine substitution at Glu(206) in TM5 abolished PMAT activity without affecting cell surface expression. Eliminating the charge at Glu(206) (E206Q) resulted in loss of organic cation transport activity, whereas conserving the negative charge (E206D) restored transporter function. Interestingly, mutant E206Q, which possesses the equivalent residue in ENT1, gained uridine transport activity. Thr(220), another residue in TM5, also showed an effect on PMAT activity. Helical wheel analysis of TM5 revealed a distinct amphipathic pattern with Glu(206) and Thr(220) clustered in the center of the hydrophilic face. In summary, our results suggest that Glu(206) functions as a critical charge sensor for cationic substrates and TM5 forms part of the substrate permeation pathway in PMAT.

SUBMITTER: Zhou M

PROVIDER: S-EPMC2672960 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Molecular determinants of substrate selectivity of a novel organic cation transporter (PMAT) in the SLC29 family.

Zhou Mingyan M Xia Li L Engel Karen K Wang Joanne J

The Journal of biological chemistry 20061122 5

Plasma membrane monoamine transporter (PMAT or ENT4) is a newly cloned transporter assigned to the equilibrative nucleoside transporter (ENT) family (SLC29). Unlike ENT1-3, PMAT mainly functions as a polyspecific organic cation transporter. In this study, we investigated the molecular mechanisms underlying the unique substrate selectivity of PMAT. By constructing chimeras between human PMAT and ENT1, we showed that a chimera consisting of transmembrane domains (TM) 1-6 of PMAT and TM7-11 of hENT ...[more]

PMID: 17121826

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Project description:Peptidase-containing ABC transporters (PCATs) are a widely distributed family of transporters which secrete double-glycine (GG) peptides. In the opportunistic pathogen Streptococcus pneumoniae (pneumococcus), the PCATs ComAB and BlpAB have been shown to secrete quorum-sensing pheromones and bacteriocins related to the competence and pneumocin pathways. Here, we describe another pneumococcal PCAT, RtgAB, encoded by the rtg locus and found intact in 17% of strains. The Rgg/SHP-like quorum-sensing system RtgR/S, which uses a peptide pheromone with a distinctive Trp-X-Trp motif, regulates expression of the rtg locus and provides a competitive fitness advantage in a mouse model of nasopharyngeal colonization. RtgAB secretes a set of coregulated rtg GG peptides. ComAB and BlpAB, which share a substrate pool, do not secrete the rtg GG peptides. Similarly, RtgAB does not efficiently secrete ComAB/BlpAB substrates. We examined the molecular determinants of substrate selectivity between ComAB, BlpAB, and RtgAB and found that the GG peptide signal sequences contain all the information necessary to direct secretion through specific transporters. Secretion through ComAB and BlpAB depends largely on the identity of four conserved hydrophobic signal sequence residues previously implicated in substrate recognition by PCATs. In contrast, a motif situated at the N-terminal end of the signal sequence, found only in rtg GG peptides, directs secretion through RtgAB. These findings illustrate the complexity in predicting substrate-PCAT pairings by demonstrating specificity that is not dictated solely by signal sequence residues previously implicated in substrate recognition.IMPORTANCE The export of peptides from the cell is a fundamental process carried out by all bacteria. One method of bacterial peptide export relies on a family of transporters called peptidase-containing ABC transporters (PCATs). PCATs export so-called GG peptides which carry out diverse functions, including cell-to-cell communication and interbacterial competition. In this work, we describe a PCAT-encoding genetic locus, rtg, in the pathogen Streptococcus pneumoniae (pneumococcus). The rtg locus is linked to increased competitive fitness advantage in a mouse model of nasopharyngeal colonization. We also describe how the rtg PCAT preferentially secretes a set of coregulated GG peptides but not GG peptides secreted by other pneumococcal PCATs. These findings illuminate a relatively understudied part of PCAT biology: how these transporters discriminate between different subsets of GG peptides. Ultimately, expanding our knowledge of PCATs will advance our understanding of the many microbial processes dependent on these transporters.

Project description:Organic cation transporters are membrane potential-dependent facilitative diffusion systems. Functional studies, extensive mutagenesis, and homology modeling indicate the following mechanism. A transporter conformation with a large outward-open cleft binds extracellular substrate, passes a state in which the substrate is occluded, turns to a conformation with an inward-open cleft, releases substrate, and subsequently turns back to the outward-open state. In the rat organic cation transporter (rOct1), voltage- and ligand-dependent movements of fluorescence-labeled cysteines were measured by voltage clamp fluorometry. For fluorescence detection, cysteine residues were introduced in extracellular parts of cleft-forming transmembrane α-helices (TMHs) 5, 8, and 11. Following expression of the mutants in Xenopus laevis oocytes, cysteines were labeled with tetramethylrhodamine-6-maleimide, and voltage-dependent conformational changes were monitored by voltage clamp fluorometry. One cysteine was introduced in the central domain of TMH 11 replacing glycine 478. This domain contains two amino acids that are involved in substrate binding and two glycine residues (Gly-477 and Gly-478) allowing for helix bending. Cys-478 could be modified with the transported substrate analog [2-(trimethylammonium)-ethyl]methanethiosulfonate but was inaccessible to tetramethylrhodamine-6-maleimide. Voltage-dependent movements at the indicator positions of TMHs 5, 8, and 11 were altered by substrate applications indicating large conformational changes during transport. The G478C exchange decreased transporter turnover and blocked voltage-dependent movements of TMHs 5 and 11. [2-(Trimethylammonium)-ethyl]methanethiosulfonate modification of Cys-478 blocked substrate binding, transport activity, and movement of TMH 8. The data suggest that Gly-478 is located within a mechanistically important hinge domain of TMH 11 in which substrate binding induces transport-related structural changes.

Dataset Information

Molecular determinants of substrate selectivity of a novel organic cation transporter (PMAT) in the SLC29 family.

Publications

Molecular determinants of substrate selectivity of a novel organic cation transporter (PMAT) in the SLC29 family.

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