Prokaryotic proteins destined for transport out of the cytoplasm typically contain an N-terminal extension sequence, called the signal peptide, which is required for export. It is evident that many secretory proteins utilize a common export system, yet the signal sequences themselves display very little primary sequence homology. In attempting to understand how different signal peptides are able to promote protein secretion through the same pathway, the physical features of natural signal sequences have been extensively examined for similarities that might play a part in function. Experimental data have confirmed statistical analyses which highlighted dominant features of natural signal sequences in Escherichia coli: a net positive charge in the N-terminus increases efficiency of transport; the core region must maintain a threshold level of hydrophobicity within a range of length limitations; the central portion adopts an alpha-helical conformation in hydrophobic environments; and the signal cleavage region is ideally six residues long, with small side-chain amino acids in the -1 and -3 positions. This review focuses on the parallels between signal peptide physical features and their functions, which emerge when the results of a variety of experimental approaches are combined. The requirement for each property may be ascribed to a potential interaction that is critical for efficient protein export. The summation of the key physical features produces signal peptides with the flexibility to function in multiple roles in order to expedite secretion. In this way, nature has indeed evolved exquisitely tuned signal sequences.

Transported proteins often contain an extension sequence called the signal peptide. The alkaline phosphatase (PhoA) signal sequence represents a typical signal peptide for comparison to idealized sequences both in vivo and in vitro. We have designed a series of idealized signal sequences which vary in amino terminal charge and core region hydrophobicity with minimal variation in amino acid composition. The idealized core regions contain different proportions of leucine and alanine residues, effectively producing hydrophobicities above and below the threshold level required for efficient secretion. The flanking amino and carboxyl termini were designed to maintain the general features and relative hydrophobicity of their counterparts in the wild-type PhoA signal sequence. Using the phoA gene, the signal peptide region was modified to generate mutants corresponding to the model sequences. Transport studies in Escherichia coli confirmed that completely idealized signal sequences, which lack a helix-breaking proline or glycine residue, can be functional if the core region is sufficiently hydrophobic and that one positively charged residue in the amino terminus is adequate for efficient transport. The corresponding peptides were chemically synthesized and exhibited HPLC retention times that reflect the relative hydrophobicities of the sequences. Structural analyses of the isolated peptides by circular dichroism demonstrate solvent dependence and exceptionally stable alpha-helix formation by the functional signal peptides in trifluoroethanol. Although leucine and alanine residues are often predicted to have similar propensities for forming an alpha-helix, considerably higher alpha-helical content is observed in the signal peptides which contain predominantly polyleucine core regions.(ABSTRACT TRUNCATED AT 250 WORDS)

We have constructed a series of signal sequence mutants that contain negatively charged amino termini and simplified core regions of varying hydrophobicity levels. This series provides a means of exploring the relative roles of the amino terminus and the hydrophobic core region during transport. The signal peptides with highly hydrophobic core regions support a rapid rate of transport in the presence of a negatively charged amino terminus. We have found that these negatively charged mutants are secreted in a manner similar to the wild-type signal sequence; sodium azide and carbonyl cyanide 3-chlorophenylhydrazone treatments indicate that the negatively charged mutants depend on SecA and the protonmotive force, respectively. These same mutants also demonstrate reduced competition with coexpressed beta-lactamase, reflecting the lower overall affinity for the transport pathway due to the net negative charge at the amino terminus. In addition, the pronounced effects of introducing three negative charges support the conclusion that the two regions function in a concerted manner.

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.