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Functional sensitivity of polar surfaces on transmembrane helix 8 and cytoplasmic loop 8-9 of the Escherichia coli GABA (4-aminobutyrate) transporter encoded by gabP: mutagenic analysis of a consensus amphipathic region found in transporters from bacteria to mammals.

ABSTRACT: The gab permease (GabP) catalyses transport of GABA (4-aminobutyrate) into Escherichia coli. Although GabP can recognize and transport many GABA analogues that exhibit activity at GABAergic synapses in the nervous system, the protein domains responsible for these transport and ligand recognition properties have not been studied. Here we report that an amphipathic domain extending through putative transmembrane helix 8 and into the adjoining cytoplasmic region (loop 8-9) contains a critical 20 residue zone within which mutagenesis of polar amino acids has a deleterious effect on [3H]GABA transport activity. This functionally important amphipathic domain is found to be highly conserved in the many APC family transporters that are homologous to GabP. And even though members of the GAT family of GABA transporters from the animal nervous system are not homologous to GabP, an analogous amphipathic structure is found in their loop 8-9 region. These results and observations suggest: (1) that the consensus amphipathic region (CAR) in the putative helix 8 and loop 8-9 region of GabP has functional significance, and (2) that nature has repeatedly used this CAR in transporters from bacteria to mammals.

SUBMITTER: Hu LA

PROVIDER: S-EPMC1219204 | biostudies-other | 1998 Mar

REPOSITORIES: biostudies-other

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Project description:One of the fundamental features of biomembranes is the ability to fuse or to separate. These processes called respectively membrane fusion and fission are central in the homeostasis of events such as those related to intracellular membrane traffic. Proteins that contain amphipathic helices (AHs) were suggested to mediate membrane fission via shallow insertion of these helices into the lipid bilayer. Here we analyze the AH-containing proteins that have been identified as essential for membrane fission and categorize them in few subfamilies, including small GTPases, Atg proteins, and proteins containing either the ENTH/ANTH- or the BAR-domain. AH-containing fission-inducing proteins may require cofactors such as additional proteins (e.g., lipid-modifying enzymes), or lipids (e.g., phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidic acid [PA], or cardiolipin). Both PA and cardiolipin possess a cone shape and a negative charge (-2) that favor the recruitment of the AHs of fission-inducing proteins. Instead, PtdIns(4,5)P2 is characterized by an high negative charge able to recruit basic residues of the AHs of fission-inducing proteins. Here we propose that the AHs of fission-inducing proteins contain sequence motifs that bind lipid cofactors; accordingly (K/R/H)(K/R/H)xx(K/R/H) is a PtdIns(4,5)P2-binding motif, (K/R)x6(F/Y) is a cardiolipin-binding motif, whereas KxK is a PA-binding motif. Following our analysis, we show that the AHs of many fission-inducing proteins possess five properties: (a) at least three basic residues on the hydrophilic side, (b) ability to oligomerize, (c) optimal (shallow) depth of insertion into the membrane, (d) positive cooperativity in membrane curvature generation, and (e) specific interaction with one of the lipids mentioned above. These lipid cofactors favor correct conformation, oligomeric state and optimal insertion depth. The most abundant lipid in a given organelle possessing high negative charge (more negative than -1) is usually the lipid cofactor in the fission event. Interestingly, naturally occurring mutations have been reported in AH-containing fission-inducing proteins and related to diseases such as centronuclear myopathy (amphiphysin 2), Charcot-Marie-Tooth disease (GDAP1), Parkinson's disease (?-synuclein). These findings add to the interest of the membrane fission process whose complete understanding will be instrumental for the elucidation of the pathogenesis of diseases involving mutations in the protein AHs.

Project description:The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776].

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Functional sensitivity of polar surfaces on transmembrane helix 8 and cytoplasmic loop 8-9 of the Escherichia coli GABA (4-aminobutyrate) transporter encoded by gabP: mutagenic analysis of a consensus amphipathic region found in transporters from bacteria to mammals.

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