PRO-OMPA DERIVATIVES WITH A HIS(6) TAG IN THEIR N-TERMINAL TRANSLOCATION INITIATION DOMAINS ARE ARRESTED BY NI2-TARGETING STAGE OF TRANSLOCATION( AT AN EARLY POST)
T. Yoshihisa et K. Ito, PRO-OMPA DERIVATIVES WITH A HIS(6) TAG IN THEIR N-TERMINAL TRANSLOCATION INITIATION DOMAINS ARE ARRESTED BY NI2-TARGETING STAGE OF TRANSLOCATION( AT AN EARLY POST), The Journal of biological chemistry, 271(16), 1996, pp. 9429-9436
We examined in vitro translocation of pro-OmpA derivatives with a His(
6) tag at various positions in their mature proteins and with a c-Myc
tag at their C termini across inverted membrane vesicles of Escherchia
coil, Those with a His(6) tag in the N-terminal region of the mature
domain, which corresponds to the ''translocation initiation domain'' p
roposed previously (Andersson, H., and von Heijne, G. (1991) Proc. Nat
l. Acad. Sci. U.S.A. 88, 9751-9754), could not be translocated in the
presence of 100 mu M Ni2+, while OmpA derivatives with a His, tag in t
he middle of or at the C terminus did not show such Ni2+ sensitivity.
The inhibitory action of Ni2+ on pro-3His-OmpA' (with a His(6) tag aft
er the third amino acid of the mature OmpA-c-Myc region) translocation
was exerted only during early events, after which it became ineffecti
ve, The inhibition point of Ni2+ was suggested to lie between membrane
targeting and exposure of the signal cleavage site to the periplasm s
ince the unprocessed and membrane-bound form of pro-3His-OmpA' was acc
umulated by the addition of Ni2+, The Ni2+-''trapped'' precursor was r
eleased from its translocation block by 30 mM histidine, which should
compete with the His, tag on the precursor protein for formation of a
Ni2+ chelating complex, We propose that Ni2+ confers a reversible posi
tive charge effect on the His(6)-tagged initiation domain of the pro-O
mpA derivatives and inhibits an early event(s) of protein translocatio
n, such as presentation of the precursor to the membranous part of the
translocase, This system will be useful in dissecting early events of
the protein translocation pathway.