Identification of a region required for binding to presecretory protein in Bacillus subtilis Ffh, a homologue of the 54-kDa subunit of mammalian signal recognition particle.
Bacillus subtilis Ffh protein is a homologue of the 54-kDa subunit of mammalian signal recognition particle (SRP54). It contains three highly hydrophobic regions (h1, h2, and h3) in the C-terminal methionine-rich domain (M-domain). Two of the hydrophobic regions, h2 and h3, are essential for small cytoplasmic RNA (scRNA) binding [Kurita, K., Honda, K., Suzuma, S., Takamatsu, H., Nakamura, K., & Yamane, K. (1996) J. Biol. Chem. 271, 13,140-13,146]. Using purified presecretory proteins and mutant Ffh proteins, we identified a region required for presecretory protein binding in B. subtilis Ffh. Deletion of this region, which consisted of residues Ser311-Gly362 of B. subtilis Ffh, including a hydrophobic sequence (h1), reduced precursor binding activity. In contrast, deletions of residues Leu121-Lys279, Lys364-Met446, or Leu338-Ser397 of B. subtilis Ffh did not. We also analyzed the mutant B. subtilis Ffh proteins, FfhQQQR and FfhQQQQ having wild-type residues 398-401 (Arg-Arg-Lys-Arg) replaced with Gln3Arg and Gln4, respectively. FfhQQQR bound to both scRNA and presecretory protein. Although the FfhQQQQ mutation prevented binding to scRNA, binding to the precursor was not affected. FfhQQQR restored the growth of B. subtilis DF46 strain in which ffh gene expression is regulated by an inducible promoter in the absence of an inducer, whereas FfhQQQQ did not. These results indicate that the region including h1 is required for B. subtilis Ffh to bind to presecretory protein. The results also suggest that scRNA is required for the complete function of the B. subtilis SRP-like particle in vivo, although this protein is intrinsically capable of binding a signal peptide free from scRNA.
Takamatsu
H
H
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Bunai
K
K
Horinaka
T
T
Oguro
A
A
Nakamura
K
K
Watabe
K
K
Yamane
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000737:chemistry* / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002455:Cell Division / Q000235:genetics; D003593:Cytoplasm / Q000235:genetics; D029968:Escherichia coli Proteins; D008322:Mammals; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D018271:Signal Recognition Particle / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D011498:Protein Precursors; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D018271:Signal Recognition Particle; D001618:beta-Lactamases