Genetic dissection of SecA: suppressor mutations against the secY205 translocase defect.
BACKGROUND
The driving force for protein translocation across the bacterial plasma membrane is provided by SecA ATPase, which undergoes striking conformational changes characterized by the membrane insertion and deinsertion cycle. This action of SecA requires the membrane-embedded SecYEG complex. Previously, we have identified a cold-sensitive secY mutation (secY205), affecting the most carboxy-terminal cytosolic domain, that did not allow an ATP-dependent insertion of a SecA-preprotein complex. Thus, this mutant provides an excellent system for genetic analysis of the SecY-SecA interaction.


RESULTS
We carried out a systematic isolation of secA mutations that suppressed secY205 cold-sensitivity. A total of 40 independent suppressor mutations were classified into: (i) allele-specific suppressors, acting only against secY205, and (ii) 'super active' suppressors, acting against almost any sec defects. The former class of mutations, presumably with specific effects on the SecY-SecA interaction, clustered in two regions close to the Walker motif A sequences of the two ATP-binding domains. The latter mutations, enhancing general SecA activities, were mostly in or around the minor ATP-binding domain.


CONCLUSIONS
The Walker motif A regions of SecA are important for the SecA-SecY interaction that leads to the SecA conformational changes required for insertion into the SecYEG channel. The minor ATP-binding domain is important for the down-regulation of SecA activities.
Matsumoto
G
G
Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
Nakatogawa
H
H
Mori
H
H
Ito
K
K
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003080:Cold Temperature; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D016153:Genes, Suppressor; D026901:Membrane Transport Proteins; D016296:Mutagenesis; D021381:Protein Transport / Q000235:genetics; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
C090796:ATP-binding protein, bacteria; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins