Processing of chimeric mammalian cytochrome b5 precursors in Escherichia coli: reaction specificity of signal peptidase and identification of an aminopeptidase in post-translocational processing.
A chimeric precursor interlinked by an arginine residue between the full-length signal sequence of alkaline phosphatase and the eukaryotic cytoplasmic cytochrome b5 was constructed. Expression of the chimeric precursor protein in Escherichia coli resulted in efficient export of spectrally authentic cytochrome b5 into the periplasm [Karim, Harding, Evans, Kaderbhai and Kaderbhai (1993) Bio/Technology 11, 612-618]. On sequencing, the apparent absence of arginine at the N-terminus of the secreted cytochrome b5 implied that the chimera was either miscleaved by signal peptidase or further processed following signal excision by an uncharacterized peptidase. The influence of the N-terminal region of cytochrome b5 on the unusual processing of the chimeric precursor was investigated by engineering a number of variant forms in which the region between Arg+1 and the mature portion of cytochrome b5 was extended and varied. Observations of the in vivo processed patterns of these variant cytochrome b5 forms exported into the periplasm revealed that the absence of arginine was due to neither miscleavage of the translocated precursor by the signal peptidase nor the nature of the early region of cytochrome b5. In fact, the selective excision of the arginine residue occurred subsequent to signal sequence deletion by an aminopeptidase which was sensitive to the metal chelator o-phenanthroline. We show that this aminopeptidase also participates in the trimming of the N-terminal arginine residue of the bacterial alkaline phosphatase to generate the three isoenzymes in the periplasm.
Harding
V
V
Department of Biochemistry, University of Wales, Penglais, Aberystwyth, Dyfed, U.K.
Karim
A
A
Kaderbhai
N
N
Jones
A
A
Evans
A
A
Kaderbhai
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D015786:Cytochromes b5 / Q000378:metabolism*; D004274:DNA, Recombinant; D010450:Endopeptidases / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008322:Mammals; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases
D004274:DNA, Recombinant; D004792:Enzyme Precursors; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D001120:Arginine; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase