TY - JOUR
T1 - Processing of Escherichia coli alkaline phosphatase
T2 - Role of the primary structure of the signal peptide cleavage region
AU - Karamyshev, Andrew L.
AU - Karamysheva, Zemphyra N.
AU - Kajava, Andrey V.
AU - Ksenzenko, Vladimir N.
AU - Nesmeyanova, Marina A.
N1 - Funding Information:
We are grateful to J. H. Miller for kindly providing us with the collection of E. coli amber suppressors, R. Fischer for providing the plasmid vector p15SK(-) and M. G. Shlyapnikov for oligonucleotide synthesis. This study was supported by the Swiss National Science Foundation (grant 7SUPJO48349), the Russian Foundation for Basic Research (grant 96-04-48048), the Russian State Scientific Program “Modern methods of biological engineering”, and was also made possible in part by an Award of the US Civilian Research and Development Foundation for the Independent States of the FSU (CRDF Award: RBI-288).
PY - 1998/4/10
Y1 - 1998/4/10
N2 - A wide range (69) of mutant Escherichia coli alkaline phosphatases with single amino acid substitutions at positions from -5 to +1 of the signal peptide were obtained for studying protein processing as a function of the primary structure of the cleavage region. Amber suppressor mutagenesis, used to create mutant proteins, included: (i) introduction of amber mutations into respective positions of the phoA gene; and (ii) expression of each mutant phoA allele in E. coli strains producing amber suppressor tRNAs specific to Ala, Cys, Gln, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser and Tyr. Most amino acid substitutions at positions -3 and -1 resulted in a complete block of protein processing. These data give new experimental support for the '-3, -1 rule'. Only Ala, Gly and Ser at position -1 allowed protein processing, and Ala provided the highest rate of processing. The results revealed the more conservative nature of the amino acids at the -1 position of signal peptides of Gram-negative bacteria as compared with those of eukaryotic organisms. Position -3 was less regular, since not only Ala, Ser and Gly, but also Leu and Cys at this position, allowed the processing. Mutations at position -4 had an insignificant effect on the processing. Surprisingly, efficient processing was provided mainly by large amino acid residues at position -2 and by middle-sized residues at position -5, indicating that the processing rate is affected by the size of amino acid residues not only at positions -1 and -3. Conformation analysis of the cleavage site taken together with the mutation and statistical data suggests an extended β-conformation of the -5 to -1 region in the signal peptidase binding pocket.
AB - A wide range (69) of mutant Escherichia coli alkaline phosphatases with single amino acid substitutions at positions from -5 to +1 of the signal peptide were obtained for studying protein processing as a function of the primary structure of the cleavage region. Amber suppressor mutagenesis, used to create mutant proteins, included: (i) introduction of amber mutations into respective positions of the phoA gene; and (ii) expression of each mutant phoA allele in E. coli strains producing amber suppressor tRNAs specific to Ala, Cys, Gln, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser and Tyr. Most amino acid substitutions at positions -3 and -1 resulted in a complete block of protein processing. These data give new experimental support for the '-3, -1 rule'. Only Ala, Gly and Ser at position -1 allowed protein processing, and Ala provided the highest rate of processing. The results revealed the more conservative nature of the amino acids at the -1 position of signal peptides of Gram-negative bacteria as compared with those of eukaryotic organisms. Position -3 was less regular, since not only Ala, Ser and Gly, but also Leu and Cys at this position, allowed the processing. Mutations at position -4 had an insignificant effect on the processing. Surprisingly, efficient processing was provided mainly by large amino acid residues at position -2 and by middle-sized residues at position -5, indicating that the processing rate is affected by the size of amino acid residues not only at positions -1 and -3. Conformation analysis of the cleavage site taken together with the mutation and statistical data suggests an extended β-conformation of the -5 to -1 region in the signal peptidase binding pocket.
KW - Alkaline phosphatase
KW - Protein processing
KW - Protein secretion
KW - Signal peptidase
KW - Signal peptide
UR - http://www.scopus.com/inward/record.url?scp=0032502929&partnerID=8YFLogxK
U2 - 10.1006/jmbi.1997.1617
DO - 10.1006/jmbi.1997.1617
M3 - Article
C2 - 9545377
AN - SCOPUS:0032502929
SN - 0022-2836
VL - 277
SP - 859
EP - 870
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 4
ER -