Signal peptidases recognize a structural feature at the cleavage site of secretory proteins.
The cloning of the gene for staphylococcal nuclease A in the pIN-III-OmpA secretion vector results in a hybrid protein which is processed by signal peptidase I, yielding an active form of the nuclease that is secreted across the cytoplasmic membrane (Takahara, M., Hibler, D., Barr, P. J., Gerlt, J. A., and Inouye, M. (1985) J. Biol. Chem. 260, 2670-2674). Using oligonucleotide-directed site-specific mutagenesis, we have constructed a set of mutants at the cleavage site area of the precursor hybrid protein designed to alter progressively the predicted secondary structure of the cleavage site. Our results show that processing becomes increasingly defective as the turn probability decreases. These results are consistent with the structural requirement that we found for the processing of lipoprotein by signal peptidase II (Inouye, S., Duffaud, G., and Inouye, M. (1986) J. Biol. Chem. 261, 10970-10975). We conclude that secretory precursor proteins have a distinct secondary structural requirement at their cleavage site for processing by signal peptidase I, as well as by signal peptidase II.
Duffaud
G
G
Department of Biochemistry, Robert Wood Johnson Medical School at Rutgers, University of Medicine and Dentistry, Piscataway, New Jersey 08854-5635.
Inouye
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D008565:Membrane Proteins; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D008565:Membrane Proteins; D011994:Recombinant Proteins; D008836:Micrococcal Nuclease; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase