Sharyn L. Rusch

Protein transport via amino-terminal targeting sequences: common themes in diverse systems (Review)

Many proteins that are synthesized in the cytoplasm of cells are ultimately found in non-cytoplasmic locations. The correct targeting and transport of proteins must occur across bacterial cell membranes, the endoplasmic reticulum membrane, and those of mitochondria and chloroplasts. One unifying feature among transported proteins in these systems is the requirement for an amino-terminal targeting signal. Although the primary sequence of targeting signals varies substantially, many patterns involving overall properties are shared. A recent surge in the identification of components of the transport apparatus from many different systems has revealed that these are also closely related. In this review we describe some of the key components of different transport systems and highlight these common features.

Is Ffh required for export of secretory proteins

In Escherichia coli, protein export from the cytoplasm may occur via the signal recognition particle (SRP)‐dependent pathway or the Sec‐dependent pathway. Membrane proteins utilize the SRP‐dependent route, whereas many secretory proteins use the cytoplasmic Sec machinery. To examine the possibility that signal peptide hydrophobicity governs which targeting route is utilized, we used a series of PhoA signal sequence mutants which vary only by incremental hydrophobicity changes. We show that depletion of SRP, but not trigger factor, affects all the mutants examined. These results suggest secretory proteins with a variety of signal sequences, as well as membrane proteins, require SRP for export.

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

Like prokaryotic Sec-dependent protein transport, chloroplasts utilize SecA. However, we observe distinctive requirements for the stimulation of chloroplast SecA ATPase activity; it is optimally stimulated in the presence of galactolipid and only a small fraction of anionic lipid and by Sec-dependent thylakoid signal peptides but not Escherichia coli signal peptides.

Juxtaposition of signal-peptide charge and core region hydrophobicity is critical for functional signal peptides

Abstract. In Escherichia coli, exported proteins are synthesized as precursors containing an amino-terminal signal peptide which directs transport through the translocase to the proper destination. We have constructed a series of signal peptide mutants, incorporating linker sequences of varying lengths between the amino-terminal charge and core region hydrophobicity, to examine the requirement for the juxtaposition of these two structural features in promoting protein transport. In vivo and in vitro analyses indicated that high transport efficiency via signal peptides with core regions of marginal hydrophobicity absolutely requires the proximity of sufficient charge.

Signal sequences containing multiple aromatic residues.

Using homopolymeric units of either phenylalanine or tryptophan to replace the natural core segment of the Escherichia coli alkaline phosphatase signal peptide, the hydrophobicity requirements for protein export and processing were further explored. The mutant signal peptide containing polyphenylalanine functioned at least as efficiently as the wild-type, while the signal incorporating polytryptophan was dysfunctional. The transport properties of these mutants confirm our work with sequences rich in aliphatic residues; namely that a high mean hydrophobicity per residue is critical for complete and rapid precursor processing and for translocation of the protein. The efficient transport properties of the polyphenylalanine-containing signal peptide demonstrate that neither the bulky, aromatic nature of phenylalanine nor the unusually high hydrophobicity of this mutant peptide adversely alters function. This study also suggests that the low occurrence of phenylalanine in natural signal sequences is not of functional consequence but probably reflects the low number of DNA codons for this residue. The polytryptophan-containing precursor was membrane inserted but not translocated. This type of transport defect suggests that this is a weakly hydrophobic signal peptide, consistent with hydropathy scales, which indicate that tryptophan is comparable to alanine. This application of polymeric sequences provides a function-based assay for the evaluation of amino acid hydrophobicity.