Crystallization of the chaperone protein SecB

Journal of Biological Chemistry, 1999

In Escherichia coli, transmembrane translocation of proteins can proceed by a number of routes. A subset of periplasmic proteins are exported via the Tat pathway to which proteins are directed by N-terminal "transfer peptides" bearing the consensus (S/T)RRXFLK "twinarginine" motif. The Tat system involves the integral membrane proteins TatA, TatB, TatC, and TatE. Of these, TatA, TatB, and TatE are homologues of the Hcf106 component of the ⌬pH-dependent protein import system of plant thylakoids. Deletion of the tatB gene alone is sufficient to block the export of seven endogenous Tat substrates, including hydrogenase-2. Complementation analysis indicates that while TatA and TatE are functionally interchangeable, the TatB protein is functionally distinct. This conclusion is supported by the observation that Helicobacter pylori tatA will complement an E. coli tatA mutant, but not a tatB mutant. Analysis of Tat component stability in various tat deletion backgrounds shows that TatC is rapidly degraded in the absence of TatB suggesting that TatC complexes, and is stabilized by, TatB.

Journal of Biological Chemistry, 2005

Journal of bacteriology, 1993

The Escherichia coli SecB protein is a cytosolic chaperone protein that is required for rapid export of a subset of exported proteins. To aid in elucidation of the activities of SecB that contribute to rapid export kinetics, mutations that partially suppressed the export defect caused by the absence of SecB were selected. One of these mutations improves protein export in the absence of SecB and is the result of a duplication of SecA coding sequences, leading to the synthesis of a large, in-frame fusion protein. Unexpectedly, this mutation conferred a second phenotype. The secA mutation exacerbated the defective protein export caused by point mutations in the signal sequence of pre-maltose-binding protein. One explanation for these results is that the mutant SecA protein has sustained a duplication of its binding site(s) for exported protein precursors so that the mutant SecA is altered in its interaction with precursor molecules.

FEBS Letters, 1989

A high‐expression plasmid for the secA gene was constructed. The SecA protein was then overproduced in E. coli and purified. The purified SecA stimulated the in vitro translocation of a model secretory protein into inverted membrane vesicles pretreated with 4 M urea. Membrane vesicles from a secAts mutant exhibited lower translocation activity, which was enhanced by SecA. These results indicate that SecA is directly involved in protein secretion across the cytoplasmic membrane.

Journal of Biological Chemistry, 1995

We have shown previously that the 100-residue-long periplasmic N-terminal tail of the Escherichia coli inner membrane protein ProW can be translocated across the inner membrane in a sec-independent manner and that its translocation is blocked by the introduction of three positively charged residues near its C-terminal end (Whitley, P., Zander, T., Ehrmann, M., Haardt, M., Bremer, E., and von Heijne, G.(1994) EMBO J. 13, 4653-4661). We have now further analyzed the requirements for translocation of the N-terminal tail and ...

The EMBO journal, 1994

The ProW protein, located in the inner membrane of Escherichia coli, has a very unusual topology with a 100-residue-long N-terminal tail protruding into the periplasmic space. We have studied the mechanism of membrane translocation of the periplasmic tail by analysing ProW-PhoA and ProW-Lep fusion proteins, both in wild-type cells and in cells with an impaired sec machinery. Our results show that the translocation efficiency is not affected by treatments that compromise the SecA and SecY functions, but that translocation is completely blocked by dissipation of the proton motive force or by the introduction of extra positively charged residues into the N-terminal tail. This suggests that the sec machinery can act properly only on domains located on the C-terminal side of a translocation signal, and that the N-terminal tail is driven through the membrane by a mechanism that involves the proton motive force.

Journal of Cell Biology, 2012

The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between...

Nature, 2007

A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.

Molecular and cellular biology, 1992

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock...

The EMBO journal, 1990

Cold-sensitive mutations in the secD locus of Escherichia coli result in severe defects in protein export at the non-permissive temperature of 23 degrees C. DNA sequence of a cloned fragment that includes the secD locus reveals open reading frames for seven polypeptide chains. Both deletions and TnphoA insertions in this clone have been used in maxicell and complementation studies to define the secD locus and its products. The secD mutations fall into two complementation groups, defining genes we have named secD and secF. These two genes comprise an operon, the first case of two genes involved in the export process being co-transcribed. The DNA sequence of the two genes along with alkaline phosphatase fusion analysis indicates that they code for integral proteins of the cytoplasmic membrane. We suggest that these two proteins may form a complex in the membrane which acts at late steps in the export process.