Substrate specificity of eukaryotic signal peptidase. Site-saturation mutagenesis at position -1 regulates cleavage between multiple sites in human pre (delta pro) apolipoprotein A-II.
We have previously described a novel mutant of human preproapolipoprotein A-II (pre(delta pro)apoA-II) in which the wild-type 18-amino acid-long signal sequence (Gly18 decreases) was functionally redefined to 20 amino acids in length (Ala 20 decreases). We have used this mutant as a model preprotein to probe the substrate specificity of eukaryotic signal peptidase. Site-saturation mutagenesis was performed resulting in the substitution of 13 different amino acids (acidic, basic, aromatic, hydrophobic, and small-neutral) for Ala20 (or position -1). The effects of these substitutions were assessed using an in vitro transcription/translation/microsomal membrane processing system. NH2-terminal sequence analysis of the 13 mutant proteins demonstrated that amino acids which occupy position -1 in a signal peptide are critical in establishing a good context for signal peptidase cleavage and that two or more potential sites of cleavage may compete for recognition by signal peptidase.
Folz
R J
RJ
Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110.
Nothwehr
S F
SF
Gordon
J I
JI
D000595:Amino Acid Sequence; D001054:Apolipoproteins A / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003062:Codon; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D001054:Apolipoproteins A; D003062:Codon; D008565:Membrane Proteins; D011498:Protein Precursors; C039768:preproapolipoprotein A-II; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase