A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex.
An approach to identifying the interaction site of multicomponent protein assembly has been applied to the membrane-bound SecY-SecE complex, which mediates protein export across the Escherichia coli cytoplasmic membrane. A dominant negative secY allele, secY-d1, inactivates SecY but preserves its ability to interact with SecE. Thus, the mutant protein sequesters SecE in an inactive complex. Second site mutations that disrupt the SecE binding site will suppress the export interference. We introduced insertion/deletion mutations that intragenically suppressed secY-d1. After eliminating knock-out mutations by virtue of the expression of a LacZ alpha sequence that had been attached to the C terminus, we obtained a striking clustering of mutations in cytoplasmic domain 4. On the basis of this result, the secY24 (Ts) substitution mutation in this domain was examined for its effects on interaction with SecE. It indeed suppressed secY-d1. Although the instability associated with excess SecY can be alleviated by overproduction of SecE, the secY24 mutant protein was not stabilized by SecE. The basal-level SecY24 protein was also destabilized at 42 degrees C. SecE was coimmunoprecipitated with SecY+ but not with the SecY24 protein. These results indicate that the secY24 mutation weakens SecY's interaction with SecE. Taken together, we propose that cytoplasmic domain 4 is important for the association between SecY and SecE.
Baba
T
T
Department of Cell Biology, Kyoto University, Japan.
Taura
T
T
Shimoike
T
T
Akiyama
Y
Y
Yoshihisa
T
T
Ito
K
K
D000483:Alleles; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D015151:Immunoblotting; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011485:Protein Binding; D017433:Protein Structure, Secondary; D000069816:SEC Translocation Channels; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli; D001616:beta-Galactosidase