Erik H. Manting
University of Groningen
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Featured researches published by Erik H. Manting.
Molecular Microbiology | 2000
Erik H. Manting; Arnold J. M. Driessen
Protein translocation across the bacterial cytoplasmic membrane has been studied extensively in Escherichia coli. The identification of the components involved and subsequent reconstitution of the purified translocation reaction have defined the minimal constituents that allowed extensive biochemical characterization of the so‐called translocase. This functional enzyme complex consists of the SecYEG integral membrane protein complex and a peripherally bound ATPase, SecA. Under translocation conditions, four SecYEG heterotrimers assemble into one large protein complex, forming a putative protein‐conducting channel. This tetrameric arrangement of SecYEG complexes and the highly dynamic SecA dimer together form a proton‐motive force‐ and ATP‐driven molecular machine that drives the stepwise translocation of targeted polypeptides across the cytoplasmic membrane. Recent findings concerning the translocase structure and mechanism of protein translocation are discussed and shine new light on controversies in the field.
The EMBO Journal | 2000
Erik H. Manting; Chris van der Does; Hervé W. Rémigy; Andreas Engel; Arnold J. M. Driessen
Translocase mediates preprotein translocation across the Escherichia coli inner membrane. It consists of the SecYEG integral membrane protein complex and the peripheral ATPase SecA. Here we show by functional assays, negative‐stain electron microscopy and mass measurements with the scanning transmission microscope that SecA recruits SecYEG complexes to form the active translocation channel. The active assembly of SecYEG has a side length of 10.5 nm and exhibits an ∼5 nm central cavity. The mass and structure of this SecYEG as well as the subunit stoichiometry of SecA and SecY in a soluble translocase–precursor complex reveal that translocase consists of the SecA homodimer and four SecYEG complexes.
Nature Structural & Molecular Biology | 2001
Arnold J. M. Driessen; Erik H. Manting; Chris van der Does
In Gram-negative bacteria, two distinct targeting routes assist in the proper localization of secreted and membrane proteins. Signal recognition particle (SRP) mainly targets ribosome-bound nascent membrane proteins, whereas SecB facilitates the targeting of periplasmic and outer membrane proteins. These routes converge at the translocase, a protein-conducting pore in the membrane that consists of the SecYEG complex associated with the peripheral ATPase, SecA. Recent structural studies of the targeting and the translocating components provide insights into how substrates are recognized and suggest a mechanism by which proteins are transported through an aqueous pore in the cytoplasmic membrane.
Journal of Biological Chemistry | 1999
Pier A. Scotti; Quido A. Valent; Erik H. Manting; Malene L. Urbanus; Arnold J. M. Driessen; Bauke Oudega; Joen Luirink
In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitrocross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitroreconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.
Molecular Microbiology | 1996
C. van der Does; T. den Blaauwen; de Janny Wit; Erik H. Manting; N.A. Groot; P. Fekkes; Arnold J. M. Driessen
SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide‐modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase,the SecY, SecE and SecG polypeptides, results in an increased level of membrane‐bound SecA. This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane‐associated form of SecA indicates that it exposes a carboxy‐terminal domain to the periplasmic face of the membrane.
Journal of Biological Chemistry | 1999
Erik H. Manting; A Kaufmann; C. van der Does; Arnold J. M. Driessen
The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.
Biochemistry | 1999
A Kaufmann; Erik H. Manting; Andreas K.J. Veenendaal; Arnold J. M. Driessen; C. van der Does
Biochemistry | 1998
C. van der Does; Erik H. Manting; A Kaufmann; M Lutz; Arnold J. M. Driessen
Journal of Bacteriology | 1999
Markus Herbort; Michael Klein; Erik H. Manting; Arnold J. M. Driessen; Roland Freudl
Journal of Bacteriology | 1997
Erik H. Manting; C. van der Does; Arnold J. M. Driessen