Functional analysis of the signal recognition particle in Escherichia coli by characterization of a temperature-sensitive ffh mutant.
The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use of E. coli strains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated. The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42 degrees C as well as show reduced growth at the permissive temperature of 30 degrees C. While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42 degrees C. It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function.
Park
Sei-Kyoung
SK
Department of Microbiology, 207 Science I Building, Iowa State University, Ames, IA 50011, USA.
Jiang
Fenglei
F
Dalbey
Ross E
RE
Phillips
Gregory J
GJ
D000483:Alleles; D001426:Bacterial Proteins / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000032:analysis; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D012329:RNA, Bacterial; D012335:RNA, Ribosomal / Q000096:biosynthesis; D018271:Signal Recognition Particle / Q000235:genetics / Q000502:physiology*; D013696:Temperature
C409968:4.5S RNA; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D018271:Signal Recognition Particle