A Derivative of Lipid A Is Involved in Signal Recognition Particle/SecYEG-dependent and -independent Membrane Integrations

Nishiyama, Kenichi; Ikegami, Ayao; Moser, Michael J.; Schiltz, Emile; Tokuda, Harukuni; Müller, Matthias

doi:10.1074/jbc.m608228200

Cited by 58 publications

(162 citation statements)

References 56 publications

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“…1C), as reported previously (26). The stimulation was dependent on the amount of MPIase used for reconstitution (Fig.…”

Section: Resultssupporting

confidence: 86%

“…MPIase is a membrane protein integrase (24,25) that drives both Sec-dependent and Sec-independent integration of membrane proteins (26). As the initial step of preprotein translocation is signal sequence insertion into a membrane, MPIase may also assist this step through a mechanism similar to that of the catalysis of membrane integration of Sec-independent substrates (25).…”

Section: Discussionmentioning

confidence: 99%

“…Even if this were the case, the side-by-side structure of SecYEG, caused by MPIase, is important for preprotein translocation, as significant stimulation of preprotein translocation is achieved with SecG inversion. In fact, MPIase is also essential for Secdependent protein integration into membranes (26). The mechanisms underlying the Sec-dependent integration remain to be clarified; that is, whether MPIase integrates Sec-dependent membrane proteins by directly interacting with them or whether only the side-by-side structure of SecYEG caused by MPIase is integration-competent.…”

Section: Discussionmentioning

confidence: 99%

“…We have determined the complete structure of MPIase and studied its structure-function relationship, and propose that MPIase is a glycolipozyme (25). As MPIase is not only involved in Sec-dependent integration but also stimulates preprotein translocation (26), it is highly suggested that MPIase interacts with SecYEG. In this article, we report that MPIase is essential for SecG inversion, modulating the dimer orientation of SecYEG.…”

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confidence: 99%

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Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation

Moser

Nagamori

Huber

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Presecretory proteins are translocated across biological membranes through protein-conducting channels such as Sec61 (eukaryotes) and SecYEG (bacteria). SecA, a translocation ATPase, pushes preproteins out with dynamic structural changes through SecYEG. SecG, a subunit of the SecYEG channel possessing two transmembrane stretches (TMs), undergoes topology inversion coupled with SecA-dependent translocation. Recently, we characterized membrane protein integrase (MPIase), a glycolipozyme involved in not only protein integration into membranes but also preprotein translocation. We report here that SecG inversion occurs only when MPIase associates with SecYEG. We also found that MPIase modulates the dimer orientation of SecYEG. Cysteine-scanning mutagenesis mapped SecG TM 2 to a relatively hydrophilic environment. The dimer formation of SecG, crosslinked at TM 2, was not observed on SecG inversion, indicating that SecYEG undergoes a dynamic structural change during preprotein translocation.

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“…1C), as reported previously (26). The stimulation was dependent on the amount of MPIase used for reconstitution (Fig.…”

Section: Resultssupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation

Moser

Nagamori

Huber

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…It has been found that without the minor E. coli lipid DAG, II Mtl can insert into phospholipid vesicles in vitro via a spontaneous unassisted integration process (Nishiyama et al, 2006). However DAG addition provides a kind of 'seal' so that II Mtl insertion is entirely dependent on the SRP and SecYEG complexes (Kawashima et al, 2008).…”

Section: Technique Nature Of Information Obtainedmentioning

confidence: 99%

Lipid dependencies, biogenesis and cytoplasmic micellar forms of integral membrane sugar transport proteins of the bacterial phosphotransferase system

Aboulwafa

Saier

2013

Microbiology

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Permeases of the prokaryotic phosphoenolpyruvate-sugar phosphotransferase system (PTS) catalyse sugar transport coupled to sugar phosphorylation. The lipid composition of a membrane determines the activities of these enzyme/transporters as well as the degree of coupling of phosphorylation to transport. We have investigated mechanisms of PTS permease biogenesis and identified cytoplasmic (soluble) forms of these integral membrane proteins. We found that the catalytic activities of the soluble forms differ from those of the membrane-embedded forms. Transport via the latter is much more sensitive to lipid composition than to phosphorylation, and some of these enzymes are much more sensitive to the lipid environment than others. While the membrane-embedded PTS permeases are always dimeric, the cytoplasmic forms are micellar, either monomeric or dimeric. Scattered published evidence suggests that other integral membrane proteins also exist in cytoplasmic micellar forms. The possible functions of cytoplasmic PTS permeases in biogenesis, intracellular sugar phosphorylation and permease storage are discussed.

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Cell‐Free Expression of G Protein–Coupled Receptors

Segers

Masure

2015

CP Protein Science

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The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes.

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A Derivative of Lipid A Is Involved in Signal Recognition Particle/SecYEG-dependent and -independent Membrane Integrations

Cited by 58 publications

References 56 publications

Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation

Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation

Lipid dependencies, biogenesis and cytoplasmic micellar forms of integral membrane sugar transport proteins of the bacterial phosphotransferase system

Cell‐Free Expression of G Protein–Coupled Receptors

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