Biomedical subjects

S L Rusch

Publications and source records attributed to S L Rusch.

6 recordsLinked to original sources

PubMed1996

The amino-terminal charge and core region hydrophobicity interdependently contribute to the function of signal sequences.

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.

Amino Acid Sequence↗

PubMed1994

Signal peptide cleavage regions. Functional limits on length and topological implications.

As a first step toward understanding the topology of the signal peptide with respect to the membrane during the protein export process, we have examined the constraints on the length of the cleavage region needed to achieve signal peptidase recognition and cleavage. Using the signal peptide of Escherichia coli alkaline phosphatase, a series of cleavage region mutants has been constructed. Variations in length were brought about by replacing the wild type cleavage region of the signal peptide with polymers of increasingly more residues. In each case, alanine residues are used exclusively in the -1 and -3 positions to provide only one viable cleavage site. Glutamine residues are used in all other positions in order to vary the length from 3 to 13 total residues. Analysis of these mutants revealed that cleavage regions ranging from 3 to 9 residues are completely and efficiently processed. The extent of processing drops substantially thereafter, with no processing observed for signal peptides with 13-residue long cleavage regions. A second mutant with a 13-residue long cleavage region was designed and analyzed to ensure that the lack of processing reflected a cleavage problem and not a translocation defect. The results are consistent with the notion that the signal peptidase active site is in close proximity to the periplasmic surface of the inner membrane and that interaction of the cleavage region with the signal peptidase probably depends on, and is constrained by, other interactions involving the signal peptide.

Alanine↗

PubMed1994

Transport of an export-defective protein by a highly hydrophobic signal peptide.

We have examined the sequence constraints on the amino-terminal region of the mature portion of alkaline phosphatase that are important for its efficient transport in Escherichia coli. Using a homopolymeric sequence of serines to replace 6 residues in this region, a transport-incompetent mutant was produced. Reintroduction of residues from the native sequence which restore charge and beta-turn potential resulted in little improvement. However, by replacing the hydrophobic core of the signal peptide with a homopolymeric series of leucines, not only was transport restored but precursor processing was more efficient than for the wild type and was insensitive to disruption of the protonmotive force. Moreover, we have titrated the signal peptide with leucine to alanine substitutions (Doud, S. K., Chou, M. M., and Kendall, D. A. (1993) Biochemistry 32, 1251-1256) and determined the minimum level of hydrophobicity necessary to achieve transport of the mutant protein. The results indicate that signal peptide hydrophobicity can completely override possible requirements for negatively charged residues and strong beta-turn forming potential in the mature protein and that the polyleucine-containing signal peptide may act as a generic signal sequence for the transport of non-native proteins in E. coli.

Alkaline Phosphatase↗

PubMed1994

Signal peptide hydrophobicity is finely tailored for function.

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.

Alanine↗

PubMed1992

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.

Alkaline Phosphatase↗

PubMedDate not supplied

Protein transport via amino-terminal targeting sequences: common themes in diverse systems.

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.

Bacteria↗