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J Biol Chem, Vol. 275, Issue 7, 4628-4634, February 18, 2000
Department of Molecular Life Science, Tokai University School of
Medicine, Isehara 259-1193, Japan
Resistance of Pseudomonas aeruginosa
to multiple species of antibiotics is largely attributable to
expression of the MexA, B-OprM efflux pump. The MexA protein is thought
to be located at the inner membrane and has been assumed to link the
xenobiotics-exporting subunit, MexB, and the outer membrane channel
protein, OprM. To verify this assumption, we analyzed membrane
anchoring and localization of the MexA protein.
n-[9,10-3H]Palmitic acid incorporation
experiments revealed that MexA was radiolabeled with palmitic acid,
suggesting that the MexA anchors the inner membrane via the fatty acid
moiety. To evaluate the role of lipid modification and inner membrane
anchoring, we substituted cysteine 24 with phenylalanine or tyrosine
and tested whether or not these mutant MexAs function properly. When
the mutant mexAs were expressed in the strain lacking chromosomal
mexA in the presence of
n-[9,10-3H]palmitic acid, we found
undetectable radiolabeling at the MexA band. These transformants
restored antibiotic resistance to the level of the wild-type strain,
indicating that lipid modification is not essential for MexA function.
These mutant strains contained both processed and unprocessed forms of
the MexA proteins. Cellular fractionation experiments revealed that an
unprocessed form of MexA anchored the inner membrane probably via an
uncleaved signal sequence, whereas the processed form was undetectable
in the membrane fraction. To assure that the lipid-free MexA
polypeptide could be unbound to the membrane, we analyzed the
two-dimensional membrane topology by the gene fusion technique. A total
of 78 mexA-blaM fusions covering the entire MexA polypeptide were
constructed, and all fusion sites were shown to be located at the
periplasm. To answer the question of whether or not membrane anchoring
is essential for the MexA function, we replaced the signal sequence of
the MexA protein with that of the azurin protein, which contains a
cleavable signal sequence but no lipid modification site. The signal
sequence of the azurin-MexA hybrid protein was properly processed and
bore the mature MexA, which was fully recovered in the soluble
fraction. The transformant, which expressed azurin-MexA hybrid protein
restored the antibiotic resistance to a level indistinguishable from
that of the wild-type strain. We concluded from these results that the
MexA protein is fully functional as expressed in the periplasmic space
without anchoring the inner membrane. This finding questioned the
assumption that the membrane fusion proteins connect the inner and
outer membranes.
Function of the Membrane Fusion Protein, MexA, of the MexA,
B-OprM Efflux Pump in Pseudomonas aeruginosa without an
Anchoring Membrane*
,
*
This study was supported in part by grants from the Ministry
of Education, Science, Sports, and Culture of Japan, the Ministry of
Health and Welfare of Japan under the Microbial Resistance Program, the
Japan Society for Promotion of Science, and the Tokai University School
of Medicine Project research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Life Science, Tokai University School of Medicine, Isehara-City, Shimokasuya 259-1193, Japan. Tel.: 81-463-93-5436; Fax:
81-463-93-5437; E-mail: yoneyama@is.icc.u-tokai.ac.jp.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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