Secretion of mutant leucine-specific binding proteins with internal deletions in Escherichia coli.
The leucine-specific binding protein, encoded by the livK gene, is located in the periplasm of E. coli. The present study is an attempt to identify intragenic regions that determine the efficiency of its secretion into the periplasm. C-terminal deletions or fusions of the livK gene to trpA (encoding the alpha subunit of tryptophan synthetase) were secreted with little loss of efficiency [1]. A series of deletions was constructed at the unique Sphl site within livK, near the 5' end of the region coding for the mature protein. Between 16 and 113 amino acids were deleted in the amino-terminal one-third of the protein. A few of these deletions were located within a few amino acids of the signal sequence processing site. Deletions extending within thirteen residues of the processing site were processed and secreted more slowly than normal. Secondary structure predictions suggested that the alpha-helical core region of the signal sequence extends into the mature protein in the case of the slow processing mutants, perhaps interfering with the recognition site for leader peptidase or other secretory components. These results suggest that the conformation around the signal processing site may be a critical factor in determining the efficiency of secretion. During the course of this study, it was found that the difference in molecular weight between precursor and mature forms of some binding protein mutants, as judged by SDS-PAGE, was much greater than could be accounted for by processing of the signal sequence. This anomalous mobility on gels, however, could be eliminated by performing SDS-PAGE in the presence of 6 M urea.
Adams
M D
MD
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Oxender
D L
DL
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D013347:Subcellular Fractions / Q000737:chemistry
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C024913:LivK protein, E coli; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals; D007930:Leucine