The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal‐recognition particle in Escherichia coli

• Published in: European journal of biochemistry Vol. 269; no. 22; pp. 5564 - 5571
• Main Authors: Adams, Hendrik, Scotti, Pier A., de Cock, Hans, Luirink, Joen, Tommassen, Jan
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.11.2002
• Subjects: Amino Acid Sequence; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; Glycine - chemistry; Kinetics; Molecular Sequence Data; Mutation; outer‐membrane protein; Plasmids - metabolism; Porins - chemistry; Porins - metabolism; Protein Biosynthesis; Protein Structure, Secondary; Protein Structure, Tertiary; Sec translocon; SecB; Signal Recognition Particle; Time Factors; Transcription, Genetic; translocation
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1046/j.1432-1033.2002.03262.x

Signal sequences often contain α‐helix‐destabilizing amino acids within the hydrophobic core. In the precursor of the Escherichia coli outer‐membrane protein PhoE, the glycine residue at position −10 (Gly−10) is thought to be responsible for the break in the α‐helix. Previously, we showed that substitution of Gly−10 by α‐helix‐promoting residues (Ala, Cys or Leu) reduced the proton‐motive force dependency of the translocation of the precursor, but the actual role of the helix breaker remained obscure. Here, we considered the possibility that extension of the α‐helical structure in the signal sequence resulting from the Gly−10 substitutions affects the targeting pathway of the precursor. Indeed, the mutations resulted in reduced dependency on SecB for targeting in vivo. In vitro cross‐linking experiments revealed that the G‐10L and G‐10C mutant PhoE precursors had a dramatically increased affinity for P48, one of the constituents of the signal‐recognition particle (SRP). Furthermore, in vitro cross‐linking experiments revealed that the G‐10L mutant protein is routed to the SecYEG translocon via the SRP pathway, the targeting pathway that is exploited by integral inner‐membrane proteins. Together, these data indicate that the helix breaker in cleavable signal sequences prevents recognition by SRP and is thereby, together with the hydrophobicity of the signal sequence, a determinant of the targeting pathway.