Structural requirements of Bacillus subtilis alpha-amylase signal peptide for efficient processing: in vivo pulse-chase experiments with mutant signal peptides.
The Bacillus subtilis alpha-amylase signal peptide consists of 33 amino acids from its translation initiation site. To analyze the structural requirements for efficient processing of the signal peptide, single and repeated Ala-X-Ala sequences and their modifications were introduced into B. subtilis alpha-amylase signal peptides of different lengths and the mature thermostable alpha-amylase. Then the cleavage positions and processing rates of the signal peptides were analyzed by the NH2-terminal amino acid sequences of the exported thermostable alpha-amylases and by in vivo pulse-chase experiments. In B. subtilis, the most efficient cleavage site was located at the peptide bond between Ala-33 and amino acid X at position 34, even though Val-X-Ala and six repeating Ala-X-Ala sequences were present around the cleavage site. However, the cleavage site was shifted to the peptide bond between Ala-31 and amino acid X when Ala-33 was deleted, and it was also shifted to Ala-35 and X when Ala-33 was replaced with Val-33. The shorter signal peptide consisting of 31 amino acids reduced the processing rate and alpha-amylase production. In contrast, those signal peptides were cleaved preferentially at the peptide bond between Ala-31 and amino acid X in Escherichia coli. In addition to the presence of an Ala residue at the -1 amino acid position, the length of the signal peptide was another important requirement for efficient processing.
Sakakibara
Y
Y
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Tsutsumi
K
K
Nakamura
K
K
Yamane
K
K
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014633:Valine / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000516:alpha-Amylases; D014633:Valine; D000409:Alanine