The Plug Domain of the SecY Protein Stabilizes the Closed State of the Translocation Channel and Maintains a Membrane Seal

Li, Weikai; Schulman, Sol; Boyd, Dana; Erlandson, Karl J.; Beckwith, Jon; Rapoport, Tom A.

doi:10.1016/j.molcel.2007.05.002

Cited by 109 publications

(140 citation statements)

References 31 publications

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“…This has been demonstrated experimentally by cysteine cross-linking of the plug with SecE outside the channel (5) and by observing spontaneous channel opening in conductance measurements (6). Accordingly, deletion of the plug domain is also found to have a prl phenotype (28,29).…”

Section: Sec61p Mutations Affect Protein Orientation In Distinct Ways-mentioning

confidence: 88%

“…Three mutations are exactly at corresponding positions: Asn 65 in E. coli SecY (prlA8914), Ile 191 (prlA200), and Ile 408 (prlA4-2), matching Arg 67 , Thr 185 , and Met 450 , respectively, in Sec61p. In addition, it has very recently been shown that deletion of the plug domain of E. coli SecY has a prl phenotype (28,29). We therefore set out to test our Sec61p mutations for a prl phenotype in yeast.…”

Section: Translocation Efficiency and Stability Of Mutantmentioning

confidence: 99%

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Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

Junne

Schwede

Goder³

et al. 2007

Journal of Biological Chemistry

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The orientation of most single-spanning membrane proteins obeys the "positive-inside rule", i.e. the flanking region of the transmembrane segment that is more positively charged remains in the cytosol. These membrane proteins are integrated by the Sec61/SecY translocon, but how their orientation is achieved is unknown. We have screened for mutations in yeast Sec61p that alter the orientation of single-spanning membrane proteins. We identified a class of mutants that are less efficient in retaining the positively charged flanking region in the cytosol. Surprisingly, these mutations are located at many different sites in the Sec61/SecY molecule, and they do not only involve charged amino acid residues. All these mutants have a prl phenotype that so far have only been seen in bacteria; they allow proteins with defective signal sequences to be translocated, likely because the Sec61p channel opens more easily. A similar correlation between topology defects and prl phenotype was also seen with previously identified yeast Sec61 mutants. Our results suggest a model in which the regulated opening of the translocon is required for the faithful orientation of membrane proteins.The Sec61/SecY translocon complex mediates translocation of proteins across and integration into the endoplasmic reticulum (ER) 3 membrane (1, 2). Proteins are targeted to this complex by hydrophobic signal sequences (3), and the signals and subsequent hydrophobic segments of the polypeptide are integrated into the bilayer as transmembrane helices. The translocon consists of Sec61␣ (Sec61p in yeast) with 10 transmembrane domains, and the single spanning proteins Sec61␤ (Sbh1p) and Sec61␥ (Sss1p), corresponding in bacteria to SecY/ SecG/SecE. The crystal structure of the SecY complex of the archaebacterium Methanococcus jannaschii (4) revealed a compact helix bundle that can form a hydrophilic channel with a lateral exit site. The central passage is blocked by a lumenal plug domain and a central constriction ring. To open, the plug needs to move out, and the ring must expand (5, 6). The crystal structure and cross-linking experiments (7) suggest that the translocation channel is formed by a single heterotrimer, although it has been proposed that upon opening of the channel two or more complexes may cooperate (8, 9).The simplest case of protein topogenesis is the orientation of a signal sequence upon entering the translocon. Cleavable signal sequences assume a N cyt /C exo orientation (cytoplasmic N and exoplasmic C terminus), whereas noncleavable signal-anchors of membrane proteins insert in N exo /C cyt or N cyt /C exo direction to translocate either their N-or their C-terminal end, respectively. Best established is the role of charged residues on either side of the hydrophobic core of the signal, which according to the "positive-inside rule" (10, 11) generally results in the more positive end to be cytosolic. Additional determinants are the folding of sequences N-terminal to an internal signal that may sterically hinder N-translocation (12) and the ...

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Section: Sec61p Mutations Affect Protein Orientation In Distinct Ways-mentioning

confidence: 88%

Section: Translocation Efficiency and Stability Of Mutantmentioning

confidence: 99%

Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

Junne

Schwede

Goder³

et al. 2007

Journal of Biological Chemistry

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“…The signal sequence promotes binding of the preprotein to a cleft of SecA formed by multiple domains, including the preprotein-binding domain and C-terminal domain (5)(6)(7)(8)(9). SecA then targets the preprotein to SecYEG at the membrane, where the signal sequence inserts into a channel formed by SecY (10)(11)(12). SecA also contains two nucleotide-binding domains with ATPase activity.…”

mentioning

confidence: 99%

Protein Export by the Mycobacterial SecA2 System Is Determined by the Preprotein Mature Domain

Feltcher

Gibbons

Ligon

et al. 2012

Journal of Bacteriology

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At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles "housekeeping" export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.

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“…It has been proposed that the inserting signal sequence of the preprotein inserts at a lipid exposed lateral gate between TM2 and TM7 whereupon the clamshell is opened through a widening of the central constriction and a displacement of the plug. The SecYEβ complex of M. jannaschii has been crystallized in an idle state in the absence of the SecA motor domain or the ribosome, and is considered as a resting state, in which the pore is tightly sealed by the central constriction comprises six hydrophobic residues and the plug domain (6). The structure of a SecA-SecYEG complex of Thermotoga maritima suggests a preopen state of the channel with a major movement of the lateral gate helices TM7, TM8, and TM5, and a partial displacement of the plug leaving a narrow gap in the lateral gate of 5 Å (7).…”

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confidence: 99%

Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules

Bonardi

Halža

Walko

et al. 2011

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

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Protein translocation in Escherichia coli is mediated by the translocase that in its minimal form consists of the protein-conducting channel SecYEG, and the motor protein, SecA. SecYEG forms a narrow pore in the membrane that allows passage of unfolded proteins only. Molecular dynamics simulations suggest that the maximal width of the central pore of SecYEG is limited to 16 Å. To access the functional size of the SecYEG pore, the precursor of outer membrane protein A was modified with rigid spherical tetraarylmethane derivatives of different diameters at a unique cysteine residue. SecYEG allowed the unrestricted passage of the precursor of outer membrane protein A conjugates carrying tetraarylmethanes with diameters up to 18 Å, whereas a 29 Å sized molecule blocked the translocation pore. Translocation of the protein-organic molecule hybrids was strictly proton motive force-dependent and occurred at a single pore. With an average diameter of an unfolded polypeptide chain of 4-6 Å, the pore accommodates structures of at least 22-24 Å, which is vastly larger than the predicted maximal width of a single pore by molecular dynamics simulations.secretion | Sec-system

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The Plug Domain of the SecY Protein Stabilizes the Closed State of the Translocation Channel and Maintains a Membrane Seal

Cited by 109 publications

References 31 publications

Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

Protein Export by the Mycobacterial SecA2 System Is Determined by the Preprotein Mature Domain

Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules

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