Signal peptide hydrophobicity is finely tailored for function

Abstract

In order to titrate the dependence of individual steps in protein transport on signal peptide hydrophobicity, we have examined a series of mutants which involve replacement of the hydrophobic core segment of the Escherichia coli alkaline phosphatase signal peptide. The core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues. Thus, a nonfunctional polyalanine-containing signal peptide is titrated with the more hydrophobic residue, leucine. Analysis of this series identified a midpoint for rapid precursor processing between alanine to leucine ratios of 6:4 and 5:5 [Doud et al. (1993): Biochemistry 32:1251-1256]. Examination of precursors that are processed more slowly indicates a lower limit of signal peptide hydrophobicity that permits membrane association and translocation. Analysis of precursors that are processed rapidly defines an intermediate range of hydrophobicity that is optimum; above this level precursors become insensitive to transport inhibitors such as sodium azide and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in parallel with substantial inhibition of beta-lactamase processing. Our data indicate that there is a surprisingly narrow range of signal peptide hydrophobicity which both supports transport of the protein to which it is attached and which does not have such a high affinity for the transport pathway that it disrupts the appropriate balance of other secreted proteins.

Competition between functional signal peptides demonstrates variation in affinity for the secretion pathway

“…We find that for signal peptides of comparable size, the extent to which the signal peptide in the second position is utilized depends solely on its hydrophobicity relative to the first. Furthermore, the pattern observed here parallels the precursor accumulation of wildtype ␤-lactamase that we observe when it is coexpressed with alkaline phosphatase carrying signal peptides of various hydrophobicity levels (36). A model for the utilization of one signal peptide or the other is shown diagrammatically in Fig.…”

“…2 and 3. The leucine-rich segments show mobility changes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (36) with the mobility of the high-molecular-weight species for WT-8L2A being consistent with the precursor species (p*). W, wholecell sample; P, periplasmic fraction.…”

“…Previous studies from our laboratory indicate that there is a correlation between the hydrophobicity of the signal peptide and the ability of the preprotein to accomplish different stages of the secretion process (8,36). Furthermore, that the affinity of the preprotein for SecA and Ffh varies depending on the hydrophobicity of the signal peptide has been indicated by azide treatment (36) and crosslinking experiments (43), respectively.…”

“…Furthermore, that the affinity of the preprotein for SecA and Ffh varies depending on the hydrophobicity of the signal peptide has been indicated by azide treatment (36) and crosslinking experiments (43), respectively. In parallel with this trend, mutants with highly hydrophobic signal peptides interfere with the secretion of wild-type ␤-lactamase, suggesting that these mutants may outcompete other preproteins for one or more components of the secretion pathway (36).…”

Insertion of the Polytopic Membrane Protein MalF Is Dependent on the Bacterial Secretion Machinery

“…However, MtlA inserted into membranes normally in the presence of reduced levels of SecA. Second, Rusch et al (18) have characterized the export of alkaline phosphatase precursors as a function of the relative hydrophobicity of their signal sequences. They found that signal sequences with very high hydrophobicity mediated precursor export that was not detectably inhibited by treatment of cells with NaN 3 .…”