Non-bilayer Lipids Stimulate the Activity of the Reconstituted Bacterial Protein Translocase

Does, Chris van der; Swaving, Jelto; Klompenburg, Wim van; Driessen, Arnold J. M.

doi:10.1074/jbc.275.4.2472

Cited by 115 publications

(110 citation statements)

References 59 publications

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“…SecY was identified after Western blotting using conventional procedures and anti-SecY antibody (1:2000), a generous gift of W. Wickner, Dartmouth Medical School. DEAE-52 purified His (6) -SecEYG was reconstituted into proteoliposomes as described (44)(45)(46).…”

Section: Protein Purification and Proteoliposome Reconstitutionmentioning

confidence: 99%

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Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

et al. 2004

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Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide-SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA. † This research was supported in part by National Institutes of Health Grant GM37639 (to D.A.K. Protein transport across, or integration into, biological membranes is a vital cellular process (1-3). Components of the Sec translocon, the membrane pore through which presecretory proteins (or membrane proteins) achieve membrane translocation (or integration), are the most conserved transport constituents throughout the three kingdoms of life (4).In Escherichia coli, the essential components of the translocase (5) include the membraneassociated form of SecA (6, 7) and the polytopic membrane proteins SecY, SecE (homologues of the mammalian Sec61α, Sec61γ, and the yeast ER 1 Sec61p, Sss1p, respectively), and SecG (8, 9); the latter three proteins form a stable trimeric SecYEG complex (10). SecA is an ATPase that powers the membrane translocation of hydrophilic polypeptides by coupling ATP hydrolysis with protein movement via concomitant SecA membrane insertion and deinsertion cycles (11,12). SecY protein has 10 transmembrane (TM1-TM10), six cytosolic (C1-C6), and five periplasmic (P1-P5) domains (13), and it forms the core of the passageway for the translocating polypeptide chain (14 (35,36). Suppressor analysis of prlA mutations using dysfunctional LamB signal peptides revealed that the suppressor mutations clustered in distinct regions, and TM7 of SecY was proposed to function in signal sequence recognition (37). Yet no investigation has focused on the direct interaction between a signal peptide and the E. coli translocon. Consequently, it remains unclear whether the nascent polypeptide chain is merely translocating in close proximity to SecY, SecY performs simply a proofreading function (37), or SecY is more intimately involved in the specific recognition of the signal peptide. Wang et al. Page 2Biochemistry. Author manuscript; available in PMC 2011 April 29. NIH-PA Author ManuscriptNIH-PA Author Manuscr...

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Section: Protein Purification and Proteoliposome Reconstitutionmentioning

confidence: 99%

“…Inverted inner membrane vesicles (IMVs) were prepared from E. coli BL21, containing pBAD22 His (6) -SecEYG, as described previously (42, 43) with minor modification. The isolation and purification of His (6) -SecEYG from IMVs was carried out essentially as described by van der Does et al (44,45 …”

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Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

et al. 2004

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“…Likewise, membrane binding of the catalytical domain of Escherichia coli leader peptidase was much higher with NBprone lipids present (8). NB-prone lipids stimulate the in vitro activity of the reconstituted integral bacterial protein translocase (9). On the other hand, anionic lipids affect several functions, such as the bilayer surface binding of many amphitropic proteins including DnaA (10), MinD (11), and CTP:phosphocholine cytidylyltransferase (12), and the correct translocation and assembly of various membrane proteins (13).…”

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Monoglucosyldiacylglycerol, a Foreign Lipid, Can Substitute for Phosphatidylethanolamine in Essential Membrane-associated Functions in Escherichia coli

Wikström

Xie²,

Bogdanov³

et al. 2004

Journal of Biological Chemistry

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The mechanisms by which lipid bilayer properties govern or influence membrane protein functions are little understood, but a liquid-crystalline state and the presence of anionic and nonbilayer (NB)-prone lipids seem important. An Escherichia coli mutant lacking the major membrane lipid phosphatidylethanolamine (NBprone) requires divalent cations for viability and cell integrity and is impaired in several membrane functions that are corrected by introduction of the "foreign" NBprone neutral glycolipid ␣-monoglucosyldiacylglycerol (MGlcDAG) synthesized by the MGlcDAG synthase from Acholeplasma laidlawii. Dependence on Mg 2؉ was reduced, and cellular yields and division malfunction were greatly improved. The increased passive membrane permeability of the mutant was not abolished, but protein-mediated osmotic stress adaptation to salts and sucrose was recovered by the presence of MGlcDAG. MGlcDAG also restored tryptophan prototrophy and active transport function of lactose permease, both critically dependent on phosphatidylethanolamine. Three mechanisms can explain the observed effects: NB-prone MGlcDAG improves the quenched lateral pressure profile across the bilayer; neutral MGlcDAG dilutes the high anionic lipid surface charge; MGlcDAG provides a neutral lipid that can hydrogen bond and/or partially ionize. The reduced dependence on Mg 2؉ and lack of correction by high monovalent salts strongly support the essential nature of the NB properties of MGlcDAG.The lipid bilayer of biological membranes acts as a permeability barrier permitting maintenance of essential ion gradients and is also the local environment for integral and peripheral membrane proteins. Important bilayer structural features are a liquid-crystalline state, an optimal length of the lipid chains, and critical fractions of anionic and nonbilayer-(NB) 1 prone lipids. Because of their small head groups the NB-prone lipids, when embedded in a bilayer, cause a curvature elastic stress of each monolayer with closer acyl chain packing (i.e. higher chain order) that changes the lateral pressure profile across the membrane (1). Increasing the proportion of NBprone lipids leads to a lower temperature for the bilayer to NB phase transition, which in many cases is fairly close to the growth temperature of organisms (2, 3). The balance of bilayer to NB-prone lipids may affect the passive membrane permeability of the bilayer (e.g. (4)), the folding and assembly of membrane proteins (5), and the function of important cellular processes such as cell division, transport, and osmotic responses. The essential character of such NB-prone lipids for cell membrane functions has been little studied.NB-prone lipids substantially affect the activity of several different types of proteins when studied in vitro. For example, a critical conformational change of transmembrane rhodopsin (6) and the activity of the interface-bound CTP:phosphocholine cytidylyltransferase (7) are modulated by variations in NBprone lipids and membrane curvature elastic stress, respectively. Likewise...

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“…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 (14,15), 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.…”

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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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Non-bilayer Lipids Stimulate the Activity of the Reconstituted Bacterial Protein Translocase

Cited by 115 publications

References 59 publications

Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

Monoglucosyldiacylglycerol, a Foreign Lipid, Can Substitute for Phosphatidylethanolamine in Essential Membrane-associated Functions in Escherichia coli

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

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