Mutational analysis of transmembrane regions 3 and 4 of SecY, a central component of protein translocase.
The SecYEG heterotrimeric membrane protein complex functions as a channel for protein translocation across the Escherichia coli cytoplasmic membrane. SecY is the central subunit of the SecYEG complex and contains 10 transmembrane segments (TM1 to TM10). Previous mutation studies suggested that TM3 and TM4 are particularly important for SecY function. To further characterize TM3 and TM4, we introduced a series of cysteine-scanning mutations into these segments. With one exception (an unstable product), all the mutant proteins complemented the cold-sensitive growth defect of the secY39 mutant. A combination of this secY mutation and the secG deletion resulted in synthetic lethality, and the TM3 and TM4 SecY cysteine substitution mutations were examined for their ability to complement this lethality. Although they were all positive for complementation, some of the complemented cells exhibited significant retardation of protein export. The substitution-sensitive residues in TM3 can be aligned to one side of the alpha-helix, and those in TM4 revealed a tendency for residues closer to the cytosolic side of the membrane to be more severely affected. Disulfide cross-linking experiments identified a specific contact point for TM3 and SecG TM2 as well as for TM4 and SecG TM1. Thus, although TM3 and TM4 do not contain any single residue that is absolutely required, they include functionally important helix surfaces and specific contact points with SecG. These results are discussed in light of the structural information available for the SecY complex.
Mori
Hiroyuki
H
Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
Shimokawa
Naomi
N
Satoh
Yasunari
Y
Ito
Koreaki
K
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C088616:SecG protein, E coli; C057841:SecY protein, E coli; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D003545:Cysteine