Topology of the phenylalanine-specific permease of Escherichia coli

Pi, Jing; Pittard, A. J.

doi:10.1128/jb.178.9.2650-2655.1996

Cited by 25 publications

(38 citation statements)

References 30 publications

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“…1) of PhePCys Ϫ were expressed from plasmids in a PE-containing (with pDD72) or PE-lacking (without pDD72) strain (AD93) of E. coli. The replacements were in putative extramembrane domains connecting TMs, as predicted by PheP-PhoA fusion analysis (23) and refined in this report by cysteine scanning analysis of the extramembrane domain (C2) connecting the P1 and P3 domains. All cysteine derivatives (including PhePCys Ϫ ) exhibited nearly the same transport activity compared with wild-type PheP in either PEcontaining or PE-lacking cells (data not shown).…”

Section: Resultsmentioning

confidence: 99%

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Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition

Zhang

Bogdanov

et al. 2003

Journal of Biological Chemistry

103

122

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Once inserted, transmembrane segments of polytopic membrane proteins are generally considered stably oriented due to the large free energy barrier to topological reorientation of adjacent extramembrane domains. However, the topology and function of the polytopic membrane protein lactose permease of Escherichia coli are dependent on the membrane phospholipid composition, revealing topological dynamics of transmembrane domains after stable membrane insertion (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). In this study, we show that the high affinity phenylalanine permease PheP shares many similarities with lactose permease. PheP assembled in a mutant of E. coli lacking phosphatidylethanolamine (PE) exhibited significantly reduced active transport function and a complete inversion in topological orientation of the N terminus and adjoining transmembrane hairpin loop compared with PheP in a PE-containing strain. Introduction of PE following the assembly of PheP triggered a reorientation of the N terminus and adjacent hairpin to their native orientation associated with regain of wild-type transport function. The reversible orientation of these secondary transport proteins in response to a change in phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly.Although considerable progress has been made in understanding the assembly of multispanning-membrane proteins (1, 2), the precise molecular events involved in the insertion, orientation, and proper formation of tertiary and quaternary structures of proteins in the membrane are not well defined. Most investigations have been focused on the role of amino acid sequence in directing the assembly of membrane proteins, whereas only a limited number of reports have addressed the effects of the native lipid environment in determining the correct insertion, folding, and topology of membrane proteins. Therefore, there is currently little understanding of, or ability to predict, how membrane protein topogenesis occurs in a given lipid environment. Whether there are constraints imposed on the topological organization of membrane proteins by phospholipid composition in addition to simply providing an amphipathic environment for maintenance of membrane protein conformation is also not clear.The most compelling evidence for a specific role for lipids in membrane protein topological organization is the requirement for phosphatidylethanolamine (PE) 1 for the proper orientation of the 12 transmembrane domains (TMs) of the lactose permease LacY of Escherichia coli (3). Assembly in the absence of PE results in a topological inversion of the N-terminal six TMs and their associated extramembrane domains. PE is required in a late step of maturation for the proper folding of the periplasmic extramembrane domain (P7) linking TMs VII and VIII (4). Proper folding of this domain is required for active (but not facilitated) transport of LacY substrates (5). The native topological org...

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Section: Resultsmentioning

confidence: 99%

“…Length of TM III-The hydropathy plot (25) of the deduced amino acid sequence of PheP (26) suggests a longer TM III than was originally predicted by PhoA fusions (23). Because of hydrophobic amino acid residues lying within the boxed region of the C2 domain (Fig.…”

Section: Fig 4 Determination Of Phep Topology In Pe-containing (A)mentioning

confidence: 99%

Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition

Zhang

Bogdanov

et al. 2003

Journal of Biological Chemistry

103

122

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“…1. The numbers at each extramembrane domain indicate the net charge (positive or negative) for each domain of GabP (outside of parentheses) (25) or PheP (inside parentheses) (35). ological organization.…”

Section: Discussionmentioning

confidence: 99%

Phospholipids as Determinants of Membrane Protein Topology

Zhang¹,

Campbell²,

King

et al. 2005

Journal of Biological Chemistry

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Evidence is accumulating that the topological organization and hence function of some membrane proteins are not solely determined by the amino acid sequence of the protein but are also influenced by the lipid composition of the membrane. The ␥-aminobutyric acid (GABA) permease (GabP) of Escherichia coli has been found in this study to be affected both topologically and kinetically by membrane lipids. Using single cysteine accessibility methods with viable E. coli strains of natural lipid composition and those lacking phosphatidylethanolamine (PE), we have shown that the N-terminal hairpin of GabP is inverted relative to the membrane in PE-lacking cells, with a hinge point in transmembrane domain III. The rate of GABA transport is reduced by more than 99% in PE-lacking cells. The Michaelis constant for GABA transport is not greatly affected nor is the dependence of transport on energy. However, "transport specificity ratio" analysis demon- Membrane lipid composition clearly affects protein structure and function, and local or temporal changes in lipid composition have been suggested as potential regulators of cellular processes (1). However, the precise changes in protein structure as a function of lipid environment, particularly in whole cells, have only recently been probed (2-4). The study of the effects of lipid composition on the topological organization of a subset of secondary solute transport proteins was investigated using a strain of Escherichia coli in which the level of the major phospholipid phosphatidylethanolamine (PE) 1 can be systematically controlled (4, 5). These studies demonstrated that although the protein sequence contains the primary topogenic signals that define transmembrane (TM) domains and topological organization of membrane proteins, membrane lipid composition is also a determinant of the orientation of TM domains with respect to the plane of the membrane.Initial studies were carried out on lactose permease (LacY), which is the most widely studied member of the large major facilitator superfamily. Major facilitator superfamily transporters are 12 TM domain spanning single component uni-, sym-, or antiporters of sugars, amino acids, drugs, and other low molecular weight solutes; LacY belongs to a six-member subfamily of oligosaccharide:H ϩ symporters (6). LacY either reconstituted in proteoliposomes containing PE or expressed in E. coli with wild type phospholipid composition carries out active transport of substrate against a concentration gradient. However, LacY in either whole cells (7) or proteoliposomes lacking PE (8, 9) only carries out energy-independent facilitated transport of substrate. The bioenergetic properties of cells and proteoliposomes are independent of the presence or absence of PE (7, 9). The level of functional LacY and total amount of LacY, as measured by the initial rate of facilitated transport and Western blotting, respectively, are also the same in both cases (7).Even more dramatic than the alteration in functional properties is the change in the organization...

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“…Furthermore, in light of the recent demonstration that CydDC transports cysteine (Pittman et al, 2002), it is notable that most prolines in the TMs of prokaryote amino acid transporters are highly conserved (Pi et al, 1998). Therefore, we decided to change the proline residues in CydD TM III (P142 and P151, Fig.…”

Section: Sequence-informed Targeting Of Residues For Mutagenesis In Tmentioning

confidence: 99%

Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly

Cruz-Ramos

Cook

et al. 2004

Microbiology

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Cytochrome bd is a respiratory quinol oxidase in Escherichia coli. Besides the structural genes (cydA and cydB) encoding the oxidase complex, the cydD and cydC genes, encoding an ABC-type transporter, are required for assembly of this oxidase. Recently, cysteine has been identified as a substrate (allocrite) that is transported from the cytoplasm by CydDC, but the mechanism of cysteine export to the periplasm and its role there remain unknown. To initiate an understanding of structure-function relationships in CydDC, its membrane topography was analysed by generating protein fusions between random and selected residues in the two polypeptides with both alkaline phosphatase and b-galactosidase. CydD and CydC are experimentally shown each to have six transmembrane segments, two major cytoplasmic loops and three minor periplasmic loops; both termini of each protein face the cytoplasm. The cydD1 allele is shown to have two point mutations (G319D, G429E) within the ATP-binding domain of CydD; either mutation alone is sufficient to cause loss or severe reduction of cytochrome bd assembly. A comparative sequence analysis prompted the targeting of residues in CydD for site-directed mutational analysis, which identified (i) the 'start' methionine residue, (ii) essential residues in the ATP-binding site (Walker sequence A) and (iii) a duplicated positively charged heptameric motif, R-G/T-L/M-X-T/V-L-R, in CydD cytoplasmic loop II. The replacement of arginines in these motifs with glycines resulted in Cyd " phenotypes; however, activity could be restored at these positions by replacing the glycine with lysine or histidine and hence returning the positive charge. The conservation of these charges in CydD-like proteins indicates functional importance. Evolutionary aspects of bacterial cyd genes are discussed. INTRODUCTIONEscherichia coli uses two membrane-bound terminal oxidases for aerobic respiration, cytochromes bo9 and bd (Gennis & Stewart, 1996). The genes encoding the cytochrome bd quinol oxidase, cydAB, are expressed both aerobically and anaerobically, but expression is maximal during aerobic stationary phase or in low oxygen environments (Cotter et al., 1990;. Consistent with this expression profile is the extraordinarily high affinity of cytochrome bd for O 2 (K m =3-8 nM; D' Mello et al., 1996). Cytochrome bd comprises two integral membrane polypeptides, subunit I (CydA, 58 kDa) and subunit II (CydB, 43 kDa). Subunit I contains haem b 558 directly involved in ubiquinol oxidation whilst two further haems, b 595 and d, in a catalytic binuclear centre are shared by both subunits (Jünemann, 1997;Borisov et al., 2001). All three haems are probably near the periplasmic side of the membrane (Osborne & Gennis, 1999). Loss of cytochrome bd causes a complex phenotype that extends beyond the lack of a functional oxidase. The Cyd 2 phenotype in E. coli includes (i) an inability to exit aerobically from stationary phase at 37 uC (Siegele et al., 1996); (ii) sensitivity to high temperature (Delaney et al., 1992), (iii) increased...

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Topology of the phenylalanine-specific permease of Escherichia coli

Cited by 25 publications

References 30 publications

Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition

Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition

Phospholipids as Determinants of Membrane Protein Topology

Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly

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