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.
Rusch
S L
SL
Department of Molecular & Cell Biology, University of Connecticut, Storrs 06269.
Kendall
D A
DA
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000737:chemistry*; D012995:Solubility; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000469:Alkaline Phosphatase