SecY alterations that impair membrane protein folding and generate a membrane stress

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/σE stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle–targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.

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2P103 Direct monitoring of membrane protein folding process during in-vitro expression by Surface Enhanced IR spectroscopy(03. Membrane proteins,Poster)

Seibutsu Butsuri ◽

10.2142/biophys.53.s176_1 ◽

2013 ◽

Vol 53 (supplement1-2) ◽

pp. S176

Author(s):

Kenichi Ataka ◽

Joachim Heberle ◽

Axel Baumann ◽

Silke Kerruth ◽

Ramona Schlesinger ◽

...

Keyword(s):

Protein Folding ◽

Membrane Proteins ◽

Ir Spectroscopy ◽

Membrane Protein ◽

In Vitro Expression ◽

Folding Process ◽

Membrane Protein Folding ◽

Surface Enhanced

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F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200402100 ◽

2004 ◽

Vol 165 (2) ◽

pp. 213-222 ◽

Cited By ~ 153

Author(s):

Martin van der Laan ◽

Philipp Bechtluft ◽

Stef Kol ◽

Nico Nouwen ◽

Arnold J.M. Driessen

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Atp Synthase ◽

Signal Recognition Particle ◽

Membrane Vesicles ◽

Membrane Insertion ◽

Subunit C ◽

Protein Insertion ◽

F1f0 Atp Synthase

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro–synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.

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The expanding role of the ER translocon in membrane protein folding

The Journal of Cell Biology ◽

10.1083/jcb.200711107 ◽

2007 ◽

Vol 179 (7) ◽

pp. 1333-1335 ◽

Cited By ~ 31

Author(s):

William R. Skach

Keyword(s):

Protein Folding ◽

Membrane Proteins ◽

Membrane Protein ◽

Functional Organization ◽

Conducting Channel ◽

Membrane Protein Folding ◽

Protein Biogenesis ◽

Insight Into ◽

Er Membrane

Eukaryotic polytopic membrane proteins are cotranslationally inserted into the ER membrane by a multisubunit protein-conducting channel called the Sec61 translocon. Although most major translocon components have been identified and reconstituted, their stoichiometry and functional organization remain unknown. This has led to speculative and sometimes conflicting models describing how multiple transmembrane (TM) segments might be oriented and integrated during nascent polytopic protein biogenesis. Kida et al. (see p. 1441 of this issue) shed new insight into this area by demonstrating that functional translocons exhibit a remarkable flexibility by simultaneously accommodating at least two hydrophilic translocating peptides that are separated by multiple hydrophobic TMs. These surprising findings support an expanded role for the translocon in membrane protein biogenesis and require reassessment of current views based on a single small functional pore.

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A growing toolbox of techniques for studying β-barrel outer membrane protein folding and biogenesis

Biochemical Society Transactions ◽

10.1042/bst20160020 ◽

2016 ◽

Vol 44 (3) ◽

pp. 802-809 ◽

Cited By ~ 9

Author(s):

Jim E. Horne ◽

Sheena E. Radford

Keyword(s):

Protein Folding ◽

Membrane Protein ◽

Outer Membrane ◽

Single Molecule ◽

Outer Membrane Protein ◽

Lipid Membrane ◽

Water Soluble ◽

Membrane Protein Folding ◽

Over 40 Years

Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed.

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A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation.

The Journal of Cell Biology ◽

10.1083/jcb.112.5.809 ◽

1991 ◽

Vol 112 (5) ◽

pp. 809-821 ◽

Cited By ~ 76

Author(s):

R N Thrift ◽

D W Andrews ◽

P Walter ◽

A E Johnson

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Transmembrane Domain ◽

Signal Sequence ◽

Secretory Protein ◽

In Vitro Translation ◽

Nascent Chain ◽

Transmembrane Sequence ◽

Er Membrane

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.

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In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways of Escherichia coli

Molecular Biology of the Cell ◽

10.1091/mbc.10.7.2163 ◽

1999 ◽

Vol 10 (7) ◽

pp. 2163-2173 ◽

Cited By ~ 109

Author(s):

Hans-Georg Koch ◽

Thomas Hengelage ◽

Christoph Neumann-Haefelin ◽

Juan MacFarlane ◽

Hedda K. Hoffschulte ◽

...

Keyword(s):

Escherichia Coli ◽

Plasma Membrane ◽

Membrane Proteins ◽

Signal Recognition Particle ◽

Membrane Vesicles ◽

Secretory Proteins ◽

Signal Recognition ◽

Free System ◽

E Coli

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coliwhich, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ΔμH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

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The trials and tribulations of membrane protein folding in vitro

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/s0005-2736(02)00714-9 ◽

2003 ◽

Vol 1610 (1) ◽

pp. 51-56 ◽

Cited By ~ 38

Author(s):

Paula J Booth

Keyword(s):

Protein Folding ◽

Membrane Protein ◽

Membrane Protein Folding

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BamA Alone Accelerates Outer Membrane Protein Folding In Vitro through a Catalytic Mechanism

Biochemistry ◽

10.1021/acs.biochem.5b00950 ◽

2015 ◽

Vol 54 (39) ◽

pp. 6009-6011 ◽

Cited By ~ 20

Author(s):

Ashlee M. Plummer ◽

Karen G. Fleming

Keyword(s):

Protein Folding ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Catalytic Mechanism ◽

Membrane Protein Folding

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Current strategies for protein production and purification enabling membrane protein structural biology

Biochemistry and Cell Biology ◽

10.1139/bcb-2015-0143 ◽

2016 ◽

Vol 94 (6) ◽

pp. 507-527 ◽

Cited By ~ 34

Author(s):

Aditya Pandey ◽

Kyungsoo Shin ◽

Robin E. Patterson ◽

Xiang-Qin Liu ◽

Jan K. Rainey

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Structural Biology ◽

Protein Production ◽

Protein Structures ◽

Data Bank ◽

In Vitro Translation ◽

Preparation Methods ◽

Natural Hosts

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10–15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

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