PubMed Health⌕ Search

Biomedical subjects

Nico Nouwen

Publications and source records attributed to Nico Nouwen.

13 recordsLinked to original sources
PubMed2006

YidC-mediated membrane insertion of assembly mutants of subunit c of the F1F0 ATPase.

YidC is a member of the OxaI family of membrane proteins that has been implicated in the membrane insertion of inner membrane proteins in Escherichia coli. We have recently demonstrated that proteoliposomes containing only YidC support both the stable membrane insertion and the oligomerization of the c subunit of the F(1)F(0) ATP synthase (F(0)c). Here we have shown that two mutants of F(0)c unable to form a functional F(1)F(0) ATPase interact with YidC, require YidC for membrane insertion, but fail to oligomerize. These data show that oligomerization is not essential for the stable YidC-dependent membrane insertion of F(0)c consistent with a function of YidC as a membrane protein insertase.

Amino Acid Substitution↗
PubMed2006

Identification of two interaction sites in SecY that are important for the functional interaction with SecA.

The motor protein SecA drives the translocation of (pre-)proteins across the SecYEG channel in the bacterial cytoplasmic membrane by nucleotide-dependent cycles of conformational changes often referred to as membrane insertion/de-insertion. Despite structural data on SecA and an archaeal homolog of SecYEG, the identity of the sites of interaction between SecA and SecYEG are unknown. Here, we show that SecA can be cross-linked to several residues in cytoplasmic loop 5 (C5) of SecY, and that SecA directly interacts with a part of transmembrane segment 4 (TMS4) of SecY that is buried in the membrane region of SecYEG. Mutagenesis of either the conserved Arg357 in C5 or Glu176 in TMS4 interferes with the catalytic activity of SecA but not with binding of SecA to SecYEG. Our data explain how conformational changes in SecA could be directly coupled to the previously proposed opening mechanism of the SecYEG channel.

Adenosine Triphosphatases↗
PubMed2006

SecA supports a constant rate of preprotein translocation.

In Escherichia coli, secretory proteins (preproteins) are translocated across the cytoplasmic membrane by the Sec system composed of a protein-conducting channel, SecYEG, and an ATP-dependent motor protein, SecA. After binding of the preprotein to SecYEG-bound SecA, cycles of ATP binding and hydrolysis by SecA are thought to drive the stepwise translocation of the preprotein across the membrane. To address how the length of a preprotein substrate affects the SecA-driven translocation process, we constructed derivatives of the precursor of the outer membrane protein A (proOmpA) with 2, 4, 6, and 8 in-tandem repeats of the periplasmic domain. With increasing polypeptide length, an increasing delay in the time before full-length translocation was observed, but the translocation rate expressed as amino acid translocation per minute remained constant. These data indicate that in the ATP-dependent reaction, SecA drives a constant rate of preprotein translocation consistent with a stepping mechanism of translocation.

Adenosine Triphosphatases↗
PubMed2006

Subunit a of cytochrome o oxidase requires both YidC and SecYEG for membrane insertion.

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that facilitate the insertion of membrane proteins. Depletion of YidC in E. coli leads to a specific defect in the functional assembly of major energy transducing complexes such as the F1F0 ATPase and cytochrome bo3 oxidase. Here we report on the in vitro reconstitution of the membrane insertion of the CyoA subunit of cytochrome bo3 oxidase. Efficient insertion of in vitro synthesized pre-CyoA into proteoliposomes requires YidC, SecYEG, and SecA and occurs independently of the proton motive force. These data demonstrate that pre-CyoA is a substrate of a novel pathway that involves both SecYEG and YidC.

Cell Membrane↗
PubMed2006

Topologically fixed SecG is fully functional.

It has been proposed that the bitopic membrane protein SecG undergoes topology inversion during translocation of (pre)proteins via SecYEG. Here we show that SecG covalently cross-linked to SecY cannot invert its topology while remaining fully functional in protein translocation. Our results strongly disfavor topology inversion of SecG during protein translocation.

Bacterial Proteins↗
PubMed2005

Covalently dimerized SecA is functional in protein translocation.

The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.

Adenosine Triphosphatases↗
PubMed2004

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

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.

Adenosine Triphosphatases↗
PubMed2003

SecYEG proteoliposomes catalyze the Deltaphi-dependent membrane insertion of FtsQ.

In Escherichia coli, the insertion of most inner membrane proteins is mediated by the Sec translocase. Ribosome-bound nascent chains of Sec-dependent inner membrane proteins are targeted to the SecYEG complex via the signal recognition particle pathway. We now demonstrate that the signal recognition particle-dependent co-translational membrane targeting and membrane insertion of FtsQ can be reconstituted with proteoliposomes containing purified SecYEG. SecA and a transmembrane electrical potential are essential for the translocation of the large periplasmic domain of FtsQ, whereas co-reconstituted YidC has an inhibitory effect. These data demonstrate that membrane protein insertion can be reconstituted with a minimal set of purified Sec components.

Adenosine Triphosphatases↗
PubMed2003

Conditional lethal mutations separate the M13 procoat and Pf3 coat functions of YidC: different YIDC structural requirements for membrane protein insertion.

Conditional lethal YidC mutants have been isolated to decipher the role of YidC in the assembly of Sec-dependent and Sec-independent membrane proteins. We now show that the membrane insertion of the Sec-independent M13 procoat-lep protein is inhibited in a short time in a temperature-sensitive mutant when shifted to the nonpermissive temperature. This provides an additional line of evidence that YidC plays a direct role in the insertion of the Sec-independent M13 procoat protein. However, in the temperature-sensitive mutant, the insertion of the Sec-independent Pf3 phage coat protein and the Sec-dependent leader peptidase were not strongly inhibited at the restricted temperatures. Conversely, using a cold-sensitive YidC strain, we find that the membrane insertion of the Sec-independent Pf3 coat protein is blocked, and the Sec-dependent leader peptidase is inhibited at the nonpermissive temperature, whereas the insertion of the M13 procoat protein is nearly normal. These data show that the YidC function for procoat and its function for Pf3 coat and possibly leader peptidase are genetically separable and suggest that the YidC structural requirements are different for the Sec-independent M13 procoat and Pf3 coat phage proteins that insert by different mechanisms.

Adenosine Triphosphatases↗
PubMed2002

SecY-SecY and SecY-SecG contacts revealed by site-specific crosslinking.

Protein translocation across the cytoplasmic membrane of Escherichia coli is mediated by the integral membrane complex SecYEG and the peripherally bound ATPase SecA. To probe the environment of the cytoplasmic domains of SecY within the SecYEG complex, we introduced single cysteine residues in each of the six cytoplasmic domains. Neighbouring SecY molecules with a single cysteine residue in cytoplasmic domains C1, C2 or C6 formed a disulfide bond upon oxidation. The presence of the disulfide bond between two C2 domains reversibly inhibited protein translocation. Chemical crosslinking showed that the C2 and C3 domains are in close proximity of SecG and chemical modification of the cysteine residue in the C5 domain with N-ethyl-maleimide or fluorescein-5-maleimide inactivates the SecYEG complex. Taken together, our data give novel insights in the interactions between subunits of the SecYEG complex and emphasise the importance of cytoplasmic domain C5 for SecY functioning.

Amino Acid Sequence↗
PubMed2002

SecDFyajC forms a heterotetrameric complex with YidC.

The Escherichia coli preprotein translocase is composed of a "preprotein conducting channel" domain that consists of the peripherally bound translocation ATPase SecA and the heterotrimeric SecYEG membrane protein complex. SecD, SecF, and YajC form another heterotrimeric complex that can associate with the SecYEG complex. YidC is an essential membrane protein that plays a role in the integration of newly synthesized membrane proteins, and has been shown to co-purify with SecYEG when all translocase components are overproduced. Here, we demonstrate that under conditions that YidC co-purifies with overproduced SecDFyajC it does not co-purify with overproduced SecYEG. Moreover, this interaction of YidC with the SecDFyajC complex is also found at chromosomal protein levels of SecD, SecF and YajC. Closer examination of the SecDFyajC-YidC complex showed that YidC binds to SecD and SecF, whereas YajC interacts only with SecF. As SecF and YajC have previously been shown to interact with SecY, we propose that these two proteins link the heterotetrameric SecDFyajC-YidC complex to the SecYEG complex.

Antigens, Bacterial↗