FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1984292994> ?p ?o ?g. }

• W1984292994 endingPage "50135" @default.
• W1984292994 startingPage "50128" @default.
• W1984292994 abstract "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. 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.Bernstein H.D. Bernstein J.M. Reddy M. Curr. Opin. Microbiol. 2000; 3: 203-209Crossref PubMed Scopus (40) Google Scholar, 2.Dalbey R.E. Chen M. Jiang F. Samuelson J.C. Curr. Opin. Cell Biol. 2000; 12: 435-442Crossref PubMed Scopus (17) Google Scholar), 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) 1The abbreviations used are: PEphosphatidylethanolamineTMtransmembrane domainAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acidMPB(+)-biotinyl,3-maleimidopropionomidyl-3,6-dioxaoctanediaminePhePCys–cysteineless PhePIPTGisopropyl-β-d-thiogalactopyranoside.1The abbreviations used are: PEphosphatidylethanolamineTMtransmembrane domainAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acidMPB(+)-biotinyl,3-maleimidopropionomidyl-3,6-dioxaoctanediaminePhePCys–cysteineless PhePIPTGisopropyl-β-d-thiogalactopyranoside. for the proper orientation of the 12 transmembrane domains (TMs) of the lactose permease LacY of Escherichia coli (3.Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar). 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.Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). Proper folding of this domain is required for active (but not facilitated) transport of LacY substrates (5.Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The native topological organization of at least the C6 cytoplasmic domain (connecting TMs VI and VII), the native conformation of the P7 domain, and the active transport function of LacY assembled in vivo in the absence of PE can be restored under the following conditions: post-assembly synthesis of PE in vivo (3.Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar), addition of PE to isolated membrane vesicles (4.Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar), or reconstitution of purified LacY into liposomes containing PE (6.Wang X. Bogdanov M. Dowhan W. EMBO J. 2002; 21: 5673-5681Crossref PubMed Scopus (91) Google Scholar). Phosphatidylserine is the only physiological lipid that can substitute for PE. However, in vitro, phosphatidylcholine supports the proper topological organization of proteoliposomal LacY, but not transport or the native conformation of the P7 domain (6.Wang X. Bogdanov M. Dowhan W. EMBO J. 2002; 21: 5673-5681Crossref PubMed Scopus (91) Google Scholar, 7.Bogdanov M. Umeda M. Dowhan W. J. Biol. Chem. 1999; 274: 12339-12345Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The minimum lipid requirement for the native conformation of the P7 domain and the function of LacY is a diacylphospholipid containing an ionizable amine head group organized in a bilayer (7.Bogdanov M. Umeda M. Dowhan W. J. Biol. Chem. 1999; 274: 12339-12345Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). phosphatidylethanolamine transmembrane domain 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (+)-biotinyl,3-maleimidopropionomidyl-3,6-dioxaoctanediamine cysteineless PheP isopropyl-β-d-thiogalactopyranoside. phosphatidylethanolamine transmembrane domain 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (+)-biotinyl,3-maleimidopropionomidyl-3,6-dioxaoctanediamine cysteineless PheP isopropyl-β-d-thiogalactopyranoside. The major objective of this study was to examine whether or not the topology of another polytopic membrane protein is reversibly affected by changes in phospholipid composition. To investigate the generality of the influence of specific lipids on the assembly of membrane proteins, we have focused on the high affinity phenylalanine permease PheP of E. coli. PheP and LacY are both electrochemical potential-driven transporters, but the former is a member of the amino acid/polyamine/organocation superfamily (8.Jack D.L. Paulsen I.T. Saier Jr., M.H. Microbiology. 2000; 146: 1797-1814Crossref PubMed Scopus (219) Google Scholar), whereas the latter belongs to the larger major facilitator superfamily (9.Saier Jr., M.H. Beatty J.T. Goffeau A. Harley K.T. Heijne W.H. Huang S.C. Jack D.L. Jahn P.S. Lew K. Liu J. Pao S.S. Paulsen I.T. Tseng T.T. Virk P.S. J. Mol. Microbiol. Biotechnol. 1999; 1: 257-279PubMed Google Scholar). Members of these families are diverse in substrate specificity (sugars, amino acids, drugs, and other low molecular mass compounds), yet show significant homology in function, sequence, and structure as well as similarities in putative topogenic amino acid residues. Many of these transporters are characterized by a 12-TM topology with both N and C termini located in the cytoplasm (see Fig. 1). The C-terminal halves of these proteins strictly follow the positive inside rule (10.von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1401) Google Scholar) for their cytoplasmic domains. However, the N-terminal halves contain acidic amino acids in the cytoplasmic extramembrane domain. A net positive charge in these domains dictates cytoplasmic disposition, but conserved single negatively charged residues are topogenic factors favoring periplasmic location if they are present in the proximity of the aqueous-membrane interface (11.Kiefer D. Hu X. Dalbey R.E. Kuhn A. EMBO J. 1997; 16: 2197-2204Crossref PubMed Scopus (68) Google Scholar, 12.Delgado-Partin V.M. Dalbey R.E. J. Biol. Chem. 1998; 273: 9927-9934Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13.Rutz C. Rosenthal W. Schulein R. J. Biol. Chem. 1999; 274: 33757-33763Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Here, we report a systematic comparative study of the TM topology of PheP assembled in PE-containing or PE-lacking E. coli cells. Our results are consistent with a topological inversion of the N-terminal (NT) first helical hairpin (NT-TM I-P1-TM II-C2) of PheP and alterations in transport properties when PheP is assembled in membranes lacking PE. The topological inversion could be reversed by addition of PE after membrane insertion and was accompanied by a restoration of normal transport function. These results, coupled with the results for LacY, support a specific role for membrane lipid composition in determining the topological organization and function of membrane proteins. Several other polytopic membrane proteins require PE for full function (14.Wilson D.M. Ottina K. Newman M.J. Tsuchiya T. Ito S. Wilson T.H. Membr. Biochem. 1985; 5: 269-290Crossref PubMed Scopus (15) Google Scholar, 15.van der Does C. Swaving J. van Klompenburg W. Driessen A.J. J. Biol. Chem. 2000; 275: 2472-2478Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), suggesting that lipid-assisted topogenesis might be a general property of a subset of such proteins. The topological flexibility of these proteins revealed by changes in phospholipid environment might be related to the conformational flexibility required for transport function or assembly. Materials—Chemicals were purchased from Sigma. Restriction enzymes were from New England Biolabs Inc. Nitrocellulose sheets (0.2 μm) for immunoblotting were purchased from Schleicher & Schüll. Radiolabeled material, peroxidase-labeled antibody, and the enhanced chemiluminescence detection (ECL) kit came from Amersham Biosciences. 4-Acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) and (+)-biotinyl,3-maleimidopropionamidyl-3,6-dioxaoctanediamine (MPB) were purchased from Molecular Probes, Inc. and Pierce Biotechnology, respectively. Site-directed polyclonal antibody against the N terminus of PheP was prepared by Zymed Laboratories, Inc. Anti-LacZ polyclonal antibody was purchased from Rockland Inc. Avidinhorseradish peroxidase was from Pierce. Lubrol was purchased from Nacalai Tesque (Kyoto, Japan). Pansorbin cells came from Calbiochem. The λDE3 lysogenization kit and plasmid pET-11a were from Novagen. Bacterial Strains, Plasmids, and Growth Conditions—Strain AD93 (pss93::kanR) was used as the host strain. This strain cannot make PE and is not viable without either plasmid pDD72 (pssA+camR and pSC101 temperature-sensitive replicon) or growth medium containing millimolar concentrations of MgCl2 (16.DeChavigny A. Heacock P.N. Dowhan W. J. Biol. Chem. 1991; 266: 5323-5332Abstract Full Text PDF PubMed Google Scholar). Strain AD93 (grown at 37 °C) lacks PE, and strain AD93/pDD72 (grown at 30 °C) retains the normal E. coli composition of phospholipids, including PE. Strain AA9256 (pss93::kanR ParaB-pssA+ ΔaraBA) (see Ref. 3.Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar for description and characteristics of this strain) was used to regulate PE synthesis. Strain AA9256(T7) was made by integration of λDE3 prophage (carrying the T7 RNA polymerase gene under lac operon control) into the chromosome of strain AA9256; the strain was used to express target genes under the control of the T7 promoter. The wild-type pheP gene and the pheP gene encoding PheP lacking cysteines (PhePCys–) were subcloned into plasmid pBR322 (EcoRI and SalI site) to produce plasmids pBR-pheP and pBRphePCys–, respectively. A series of pBR322-derived plasmids encoding derivatives of PhePCys– containing single Cys replacements in extramembrane domains were constructed by site-directed mutagenesis (17.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6809) Google Scholar). The nomenclature for these plasmids is pBRpheP-X, e.g. pBRpheP-G13 has a G13C replacement (Fig. 1). pETpheP-X plasmids were constructed from pET-11a (carrying lacI) by cloning pheP-X genes into the BamHI and NdeI regions, thus placing the genes under tandem T7 promoter and lac operator control. Cells were grown in LB medium containing ampicillin (100 μg/ml) as required and 50 mm MgCl2. Expression of pssA under the control of the araB promoter was either induced by growth in the presence of 0.2% arabinose or repressed by growth in the presence of 2% glucose; to maintain controlled expression, cells were maintained in exponential growth phase. Chemical Labeling of Cys Residues—A 50-ml culture of cells expressing a PheP-X derivative was grown in LB medium plus Mg2+ and ampicillin to mid-log phase (A600 = 0.5–0.6) and resuspended after harvesting to A600 = 25 in 1 ml of buffer A (100 mm K+-HEPES (pH 7.5), 250 mm sucrose, 50 mm MgCl2, and 0.1 mm KCl). Samples were incubated with 5 mm AMS for 30 min at 25 °C to block water-accessible Cys residues exposed to the periplasmic side of the cytoplasmic membrane. AMS was removed by two cycles of centrifugation and resuspension in buffer A. Cells (either with or without AMS pretreatment) were biotinylated by adding MPB to a final concentration of 100 μm, followed by incubation for 5 min at 25 °C. The reaction was quenched by addition of β-mercaptoethanol to 20 mm, followed by two cycles of centrifugation and resuspension in buffer A containing 20 mm β-mercaptoethanol. To expose Cys residues facing the cytoplasm to external solvent, cell suspensions were vortexed vigorously with 0.5% (v/v) toluene for 1 min and subjected to labeling as described above. Labeled cells were resuspended in 0.5 ml of 10 mm Tris-HCl (pH 8.0), 5 mm EDTA, and 5 mm β-mercaptoethanol and then solubilized and immunoprecipitated as described below. Immunoprecipitation and Western Blot Analysis—After MPB labeling, cells were lysed by addition of an equal volume (0.5 ml) of 0.2 m NaOH, vortexed, incubated for 15 min on ice, and then centrifuged at 20,800 × g for 15 min at 4 °C. The pellets were washed once with 4 m KCl and once with 10 mm Tris-HCl (pH 8.0) and then solubilized by resuspension in 50 μl of 50 mm Tris-HCl (pH 8.0), 1 mm EDTA, and 1% SDS, followed by vigorous vortexing for 30 min at room temperature and incubation for 10 min at 37 °C. Samples were diluted by adding 1.4 ml of cold 50 mm Tris-HCl (pH 8.0) containing 150 mm NaCl, 0.1 mm EDTA, and 0.1% Lubrol (immunoprecipitation buffer) and immunoprecipitated with anti-PheP polyclonal antibody overnight at 4 °C. The antibody complex was isolated by addition of 50 μl of a suspension of formalin-treated Staphylococcus aureus cells (Pansorbin) reconstituted in immunoprecipitation buffer according to the supplier's instructions. The samples were gently rocked at 4 °C for 60 min. After centrifugation, the pellet was washed once with immunoprecipitation buffer, twice with 1 ml of 50 mm Tris-HCl (pH 8.0) containing 1 m NaCl and 0.1% Lubrol, and once with 10 mm Tris-HCl (pH 8.0). The final precipitates were solubilized by resuspension in 30 μl of SDS sample buffer and incubated at 37 °C for 15 min. Samples were centrifuged, and the solubilized proteins were subjected to SDS-PAGE on 10–12% gels. The samples were transferred from the gels to nitrocellulose membranes as described previously (5.Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Avidin-horseradish peroxidase (1:50,000 dilution of a 2 mg/ml stock solution) and conventional ECL reagents were used to visualize biotinylated proteins according to the manufacturers' instructions. A Bio-Rad Fluor-S Max Multimager was used to record the results. Other Methods—Transport of [3H]phenylalanine was measured in intact E. coli cells as previously described (18.Wookey P.J. Pittard J. Forrest S.M. Davidson B.E. J. Bacteriol. 1984; 160: 169-174Crossref PubMed Google Scholar). Phospholipid composition of cell membranes was analyzed after 32PO4 labeling of cells as previously described (16.DeChavigny A. Heacock P.N. Dowhan W. J. Biol. Chem. 1991; 266: 5323-5332Abstract Full Text PDF PubMed Google Scholar) using chloroform/methanol/acetic acid (65: 25:9, v/v) as the chromatography solvent. Activity and Expression of PheP—Plasmid pBRpheP (wild-type pheP gene subcloned into pBR322) was transformed into PE-containing strain AD93/pDD72, which was converted to PE-lacking strain AD93 by inducing loss of pDD72 (16.DeChavigny A. Heacock P.N. Dowhan W. J. Biol. Chem. 1991; 266: 5323-5332Abstract Full Text PDF PubMed Google Scholar). The phospholipid composition of all strains was verified before and after transformation (data not shown). PheP activity was measured by the ability to actively accumulate phenylalanine against a concentration gradient. The initial rate and the steady-state level of phenylalanine transport were severely inhibited in PE-lacking cells compared with PE-containing cells (Fig. 2A). Transport was also sensitive to addition of an uncoupler, verifying that active transport was occurring in both cell types. More detailed kinetic analysis showed that both the Km (34.8 versus 6.4 μm) and Vmax (4.2 versus 21.6 nmol/min/mg) were adversely affected in PE-lacking versus PE-containing cells, respectively (Fig. 2B). However, the PheP expression levels in the membranes of PE-containing and PE-lacking cells, as measured by Western blotting with anti-PheP polyclonal antibody, were nearly the same (Fig. 2C), consistent with a change in Km. Therefore, PE-lacking E. coli membranes contain the same amount of PheP, but with impaired Phe transport function. The level of PheP resulting from the chromosomal pheP gene (Fig. 2C, first lane) was barely detectable relative to the detection of gene product from the plasmidborne copy of pheP (second lane). Rationale and Methodology for Determination of Transmembrane Topology—The topology of putative hydrophilic loops connecting TMs of PheP was determined based on the accessibility in whole cells of single cysteine residues in these loops to sulfhydryl reagents. MPB is a biotinylated sulfhydryl-specific probe that readily passes through the pores of the outer membrane, but is relatively impermeable to the inner membrane (19.Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 20.Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Therefore, cysteines located on the periplasmic side of the inner membrane should be derivatized by MPB, whereas cysteines located on cytoplasmic side of the inner membrane should be protected (3.Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar, 21.Wada T. Long J.C. Zhang D. Vik S.B. J. Biol. Chem. 1999; 274: 17353-17357Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) unless the membrane is first permeabilized with toluene (22.Jackson R.W. DeMoss J.A. J. Bacteriol. 1965; 90: 1420-1425Crossref PubMed Google Scholar). At high concentrations, some MPB does partition into the membrane and also enters the cytoplasm. To verify that MPB-derivatized cysteines were exposed to the outer surface of the inner membrane, highly hydrophilic AMS was used to block such residues prior to MPB treatment (20.Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The mildest conditions were established for each host strain as to the level of MPB that allows sufficient derivatization of periplasmic cysteines without significant reaction with cytoplasmic cysteines. Control experiments testing for biotinylation of cytoplasmic β-galactosidase (LacZ) (Fig. 3A), which contains many natural cysteine residues, demonstrated that the host cells used in this study were neither leaky nor permeable to MPB (Fig. 3B, second and fourth lanes) under the labeling conditions we chose for PheP unless the cells were first permeabilized with toluene (first and third lanes). Orientation of PheP Assembled in PE-containing and PE-lacking Cells—To assess the effect of membrane phospholipid composition on the topological organization of PheP, single Cys replacement derivatives (Fig. 1) of PhePCys– 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.Pi J. Pittard A.J. J. Bacteriol. 1996; 178: 2650-2655Crossref PubMed Google Scholar) 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–) exhibited nearly the same transport activity compared with wild-type PheP in either PE-containing or PE-lacking cells (data not shown). Western blot analysis using PheP-specific antibody showed that all these derivatives were present in the membrane fraction and at nearly the same level as wild-type PheP expressed from pBR322 (Fig. 4, A and B, lower panels). The level of endogenous PheP produced from the chromosomal copy of pheP could not be detected by either Western blotting or analysis of biotinylation at the level of protein subjected to analysis (Fig. 4, A and B, first lanes). The predicted biotinylation patterns (Fig. 4A, upper panel) were observed for single Cys derivatives (diagramed in Fig. 1) of PheP expressed and probed in PE-containing cells, i.e. only the derivatives with single cysteines in the periplasmic (P) domains were labeled, whereas the other five derivatives with single cysteines in the cytoplasmic (C) domains and the N terminus (NT) were protected from labeling. However, in PE-lacking cells (Fig. 4B, upper panel), the cysteine residues within the NT and C2 domains, which were not labeled in PE-containing cells, were strongly labeled in PE-lacking cells. Moreover, the cysteine located within the P1 loop that was fully biotinylated in PE-containing cells was not accessible in PE-lacking cells. The remainder of PheP downstream of the first helical hairpin exhibited a normal topological disposition as demonstrated by the same biotinylation patterns in PE-containing and PE-lacking cells. The Western blots (Fig. 4, A and B, lower panels) showed that the absence of a signal was not due to absence of PheP. No labeling of PheP was detected when the host strain (either +PE or –PE) expressing PhePCys– was probed, even though Western blot analysis showed that the protein was synthesized and inserted into the membrane. These results are an accurate representation of at least three experiments with each domain. To further substantiate the misorganization of the N-terminal helical hairpin (NT-TM I-P1-TM II-C2) of PheP in PE-lacking E. coli cells, single Cys derivatives expressed in both cell types were pretreated with side-specific control reagents (toluene or AMS) before MPB labeling. In PE-containing cells, when samples were permeabilized with toluene prior to MPB treatment, no additional biotinylation occurred for the periplasmic domains, but extensive biotinylation of the cytoplasmic domains occurred (Fig. 5A, left two lanes). These results are in agreement with the putative location of single cysteines in the current topology map of PheP (Fig. 1) (24.Pi J. Chow H. Pittard A.J. J. Bacteriol. 2002; 184: 5842-5847Crossref PubMed Scopus (12) Google Scholar). In PE-lacking cells, the P1 domain was rendered accessible to MPB after toluene treatment, whereas the NT and C2 domains showed no increase in accessibility, consistent with an inversion in the topological orientation of the first two TMs. The remainder of the domains behaved the same as in PE-containing cells (Fig. 5A, right two lanes). AMS is a hydrophilic, membrane-impermeable, non-biotinylated sulfhydryl reagent (20.Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Periplasmic cysteines accessible to MPB in PE-containing and PE-lacking whole cells should be blocked from reaction with MPB if the cells are pretreated with AMS; however, cysteines that lie within the bilayer and that react with bilayer-partitioned MPB would not be protected by AMS. Fig. 5B shows that, in PE-lacking (but not PE-containing) cells, the NT and C2 domains were protected by AMS, as were the P3 and P5 domains in both cell types. This further establishes that, under the conditions used, MPB did not react with cysteines sequestered in either the membrane bilayer or the lumen. A Reversible Topological Switch Modulated by a Change in Phospholipid Composition—The potential reversible nature of the inverted topology observed in PE-lacking cells was addressed to better understand the molecular basis for the perturbations in the assembly and stability of this protein. In strain AA9256(T7), the chromosomal pssA gene is under tight regulation of the araB promoter, and the synthesis of PE can be induced by growth in the presence of arabinose or can be repressed by growth on glucose (3.Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar). The pETpheP-X plasmids contain pheP genes under the control of the T7 promoter and lac operator. The T7 RNA polymerase expressed from the chromosome is also under the control of the lac operon, thereby allowing induction of PheP derivatives strictly dependent on addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to the growth medium. Cells were grown first on glucose and IPTG to allow synthesis and membrane assembly of PheP in the near absence of PE. Then, glucose and IPTG were removed, and cells were grown on arabinose to allow new synthesis of PE in the absence of newly synthesized PheP. Cells were isolated and assayed for transport function and Cys accessibility. Parallel cultures were grown with 32PO4 to determine the respective phospholipid composition. The level of PE was <10% of total phospholipids in cells grown on glucose with IPTG (Fig. 6A, left lane). PheP transport function was also reduced as expected (Fig. 6B). Treatment of intact cells expressing single Cys derivatives of PheP with MPB showed that topological misorganization of the first hairpin (NT-TM I-P1-TM II-C2) occurred (Fig. 6C), i.e. the NT, C2, and P3 domains were biotinylated with and without toluene treatment, indicating their periplasmic orientation, whereas the P1 domain was not accessible to MPB unless cells were first treated with toluene, indicating its cytoplasmic orientation. Switching to growth on arabinose in the absence of IPTG returned the phospholipid composition to normal (∼75% PE) after 90 min (Fig. 6A, right lane). Simultaneously, PheP transport function was restored (Fig. 6B), and proper topological organization was detected (Fig. 6C), i.e. the NT and C2 domains were no longer biotinylated unless cells were pretreated with toluene, whereas the P1 and P3 domains were accessible to MPB to the same extent with and without toluene. These results strongly support a reversible topological organization of the N-terminal first helical hairpin of PheP that is determined by the membrane phospholipid composition post-insertion. To establish that the restoration of PheP activity and the changes in PheP biotinylation patterns observed after switching growth from glucose to arabinose were not due to read-through expression of newly synthesized PheP in the absence of IPTG, protein radiolabeling (Fig. 6D) was performed under the same growth conditions as described above. The results indicate that there was no detectable PheP expressed during growth in the presence of arabinose without IPTG compared with the parallel positive control (induced with IPTG). Therefore, the recovered function and labeling pattern of PheP after induction of PE synthesis were due to existing PheP synthesized prior to induction of newly synthesized PE. Length of TM III—The hydropathy plot (25.Engelman D.M. Steitz T.A. Goldman A. Annu. Rev. Biophys. Biophys. Chem. 1986; 15: 321-353Crossref Pu" @default.
• W1984292994 created "2016-06-24" @default.
• W1984292994 creator A5005928599 @default.
• W1984292994 creator A5035453308 @default.
• W1984292994 creator A5074678729 @default.
• W1984292994 creator A5076699095 @default.
• W1984292994 creator A5086963474 @default.
• W1984292994 date "2003-12-01" @default.
• W1984292994 modified "2023-10-17" @default.
• W1984292994 title "Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition" @default.
• W1984292994 cites W1529350605 @default.
• W1984292994 cites W1555008776 @default.
• W1984292994 cites W1640690355 @default.
• W1984292994 cites W1761200347 @default.
• W1984292994 cites W1866602906 @default.
• W1984292994 cites W1953872558 @default.
• W1984292994 cites W1965918112 @default.
• W1984292994 cites W1966610845 @default.
• W1984292994 cites W1967167673 @default.
• W1984292994 cites W1968311587 @default.
• W1984292994 cites W1984937473 @default.
• W1984292994 cites W1985386403 @default.
• W1984292994 cites W1990499235 @default.
• W1984292994 cites W2006677268 @default.
• W1984292994 cites W2010762855 @default.
• W1984292994 cites W2011644828 @default.
• W1984292994 cites W2015432180 @default.
• W1984292994 cites W2017498808 @default.
• W1984292994 cites W2019113077 @default.
• W1984292994 cites W2024112534 @default.
• W1984292994 cites W2025891680 @default.
• W1984292994 cites W2035172570 @default.
• W1984292994 cites W2036031058 @default.
• W1984292994 cites W2039511230 @default.
• W1984292994 cites W2047055618 @default.
• W1984292994 cites W2048002152 @default.
• W1984292994 cites W2056405723 @default.
• W1984292994 cites W2056882263 @default.
• W1984292994 cites W2060913542 @default.
• W1984292994 cites W2068082270 @default.
• W1984292994 cites W2076066586 @default.
• W1984292994 cites W2087458839 @default.
• W1984292994 cites W2089095485 @default.
• W1984292994 cites W2095451791 @default.
• W1984292994 cites W2099384461 @default.
• W1984292994 cites W2106207499 @default.
• W1984292994 cites W2108645448 @default.
• W1984292994 cites W2113301046 @default.
• W1984292994 cites W2115670301 @default.
• W1984292994 cites W2120684043 @default.
• W1984292994 cites W2122040451 @default.
• W1984292994 cites W2135874673 @default.
• W1984292994 cites W2136410079 @default.
• W1984292994 cites W2140974099 @default.
• W1984292994 cites W2144112248 @default.
• W1984292994 cites W2149026054 @default.
• W1984292994 cites W2155293971 @default.
• W1984292994 cites W2160535028 @default.
• W1984292994 cites W2210371060 @default.
• W1984292994 cites W2577338546 @default.
• W1984292994 cites W262216995 @default.
• W1984292994 doi "https://doi.org/10.1074/jbc.m309840200" @default.
• W1984292994 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14525982" @default.
• W1984292994 hasPublicationYear "2003" @default.
• W1984292994 type Work @default.
• W1984292994 sameAs 1984292994 @default.
• W1984292994 citedByCount "106" @default.
• W1984292994 countsByYear W19842929942012 @default.
• W1984292994 countsByYear W19842929942013 @default.
• W1984292994 countsByYear W19842929942014 @default.
• W1984292994 countsByYear W19842929942015 @default.
• W1984292994 countsByYear W19842929942016 @default.
• W1984292994 countsByYear W19842929942017 @default.
• W1984292994 countsByYear W19842929942018 @default.
• W1984292994 countsByYear W19842929942019 @default.
• W1984292994 countsByYear W19842929942020 @default.
• W1984292994 countsByYear W19842929942021 @default.
• W1984292994 countsByYear W19842929942022 @default.
• W1984292994 countsByYear W19842929942023 @default.
• W1984292994 crossrefType "journal-article" @default.
• W1984292994 hasAuthorship W1984292994A5005928599 @default.
• W1984292994 hasAuthorship W1984292994A5035453308 @default.
• W1984292994 hasAuthorship W1984292994A5074678729 @default.
• W1984292994 hasAuthorship W1984292994A5076699095 @default.
• W1984292994 hasAuthorship W1984292994A5086963474 @default.
• W1984292994 hasBestOaLocation W19842929941 @default.
• W1984292994 hasConcept C12554922 @default.
• W1984292994 hasConcept C138885662 @default.
• W1984292994 hasConcept C185592680 @default.
• W1984292994 hasConcept C2778918659 @default.
• W1984292994 hasConcept C40231798 @default.
• W1984292994 hasConcept C41625074 @default.
• W1984292994 hasConcept C41895202 @default.
• W1984292994 hasConcept C55493867 @default.
• W1984292994 hasConcept C86803240 @default.
• W1984292994 hasConceptScore W1984292994C12554922 @default.
• W1984292994 hasConceptScore W1984292994C138885662 @default.
• W1984292994 hasConceptScore W1984292994C185592680 @default.

• next