Organophosphate hydrolase in Brevundimonas diminuta is targeted to the periplasmic face of the inner membrane by the twin arginine translocation pathway.
A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of Brevundimonas diminuta. Expression of the precursor form of OPH carrying a C-terminal His tag in an opd-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type opd gene. Amino acid substitutions in the invariant arginine residues of the Tat signal peptide affected both the processing and localization of OPH, confirming a critical role for the Tat system in membrane targeting of OPH in B. diminuta. The localization of OPH to the periplasmic face of the inner membrane in B. diminuta was demonstrated by proteinase K treatment of spheroplasts and also by fluorescence-activated cell sorting analysis of cells expressing OPH-green fluorescent protein fusions with and without an SsrA tag that targets cytoplasmic proteins to the ClpXP protease.
Gorla
Purushotham
P
Dept. of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India.
Pandey
Jay Prakash
JP
Parthasarathy
Sunil
S
Merrick
Mike
M
Siddavattam
Dayananda
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D042063:Caulobacteraceae / Q000201:enzymology*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D007425:Intracellular Membranes / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D021381:Protein Transport
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases
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