Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M13 procoat protein by the Escherichia coli leader peptidase.
Leader peptidase cleaves the leader sequence from the amino terminus of newly made membrane and secreted proteins after they have translocated across the membrane. Analysis of a large number of leader sequences has shown that there is a characteristic pattern of small apolar residues at -1 and -3 (with respect to the cleavage site) and a helix-breaking residue adjacent to the central apolar core in the region -4 to -6. The conserved sequence pattern of small amino acids at -1 and -3 around the cleavage site most likely represents the substrate specificity of leader peptidase. We have tested this by generating 60 different mutations in the +1 to -6 domain of the M13 procoat protein. These mutants were analyzed for in vivo and in vitro processing, as well as for protein insertion into the cytoplasmic membrane. We find that in vivo leader peptidase was able to process procoat with an alanine, a serine, a glycine, or a proline residue at -1 and with a serine, a glycine, a threonine, a valine, or a leucine residue at -3. All other alterations at these sites were not processed, in accordance with predictions based on the conserved features of leader peptides. Except for proline and threonine at +1, all other residues at this position were processed by leader peptidase. None of the mutations at -2, -4, or -5 of procoat (apart from proline at -4) completely abolished leader peptidase cleavage in vivo although there were large effects on the kinetics of processing.(ABSTRACT TRUNCATED AT 250 WORDS)
Shen
L M
LM
Department of Chemistry, Ohio State University, Columbus 43210.
Lee
J I
JI
Cheng
S Y
SY
Jutte
H
H
Kuhn
A
A
Dalbey
R E
RE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000378:metabolism; D003090:Coliphages / Q000235:genetics*; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D014644:Genetic Variation; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010449:Peptide Mapping; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase