In vivo analyses of interactions between SecE and SecY, core components of the Escherichia coli protein translocation machinery.
We have carried out structure-function studies on the cytoplasmic membrane protein, SecE, a component of the Escherichia coli secretion machinery. SecE, along with SecY, form a complex in the cytoplasmic membrane essential for protein translocation. By directed mutagenesis, we altered highly conserved residues of the second cytoplasmic domain (CD2) and of the COOH-terminal periplasmic region (PD2) of SecE. These mutants, as well as previously constructed mutations in the third membrane-spanning segment of SecE (MSS3), were tested for their ability to complement a secE null mutation, for their effects on protein export in vivo, and for their ability to form a stable complex with SecY. Most single mutations at the conserved positions in CD2 caused secretion defects, but had little effect on growth at 37 degrees C. Double mutations in CD2, or the introduction or removal of proline residues, affected growth and protein translocation more severely. Co-immunoprecipitations of SecE and SecY revealed that all mutant proteins, except those altered in PD2, destabilized the SecE-SecY complex. These results suggest that several regions contribute to the formation of a stable SecE-SecY complex but the elimination of a single contact point does not necessarily affect the functionality of the complex.
Pohlschröder
M
M
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Murphy
C
C
Beckwith
J
J
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli