Requirement for the COOH-terminal pro-sequence in the translocation of aqualysin I across the cytoplasmic membrane in Escherichia coli.
Aqualysin I from Thermus aquaticus YT-1 is an extracellular subtilisin-type serine protease. The protease is synthesized as a distinct precursor composed of four functional domains: an N-terminal signal sequence, an N-terminal pro-sequence, a protease domain, and a C-terminal pro-sequence. The N-terminal pro-sequence is essential for the production of active aqualysin I while the C-terminal pro-sequence is required for extracellular secretion of aqualysin I. In an E. coli expression system, the function of C-terminal pro-sequence in the translocation of aqualysin I across the cytoplasmic membrane was investigated. More than 60-70% of the total activity was detected in the cytoplasmic fraction in the deletion mutations of the C-terminal pro-sequence while less than 30% was found in this fraction in wild type. In addition, in vitro processing of aqualysin I precursors with these mutations to a mature form promptly occurred and the folding into active aqualysin I was rapid. These results suggest that the C-terminal pro-sequence, probably in conjunction with the signal sequence, facilitates the translocation of the precursor across the cytoplasmic membrane by preventing the precursor from taking on an active conformation.
Kim
D W
DW
Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113, Japan. dong-wook.kim@yale.edu
Matsuzawa
H
H
D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology*
D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C055102:aqualysin I