Biomedical subjects

Eli O van der Sluis

Publications and source records attributed to Eli O van der Sluis.

5 recordsLinked to original sources

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

Stepwise evolution of the Sec machinery in Proteobacteria.

The Sec machinery facilitates the translocation of proteins across and into biological membranes. In several of the Proteobacteria, this machinery contains accessory features that are not present in any other bacterial division. The genomic distribution of these features in the context of bacterial phylogeny suggests that the Sec machinery has evolved in discrete steps. The canonical Sec machinery was initially supplemented with SecB; subsequently, SecE was extended with two transmembrane segments and, finally, SecM was introduced. The Sec machinery of Escherichia coli and other Enterobacteriales represents the end product of this stepwise evolution.

Adenosine Triphosphatases↗

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↗

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↗