(Received for publication, October 11, 1994 )
From the
The F sector of the Escherichia coli H
transporting ATP synthase is composed of a
complex of three subunits, each of which traverses the inner membrane.
We have studied the interdependence of subunit insertion into the
membrane in a series of chromosomal mutants in which the primary
mutation prevented insertion of one of the F
subunits.
Subunit insertion was assessed using Western blots of mutant membrane
preparations. Subunit b and subunit c were found to
insert into the membrane independently of the other two F
subunits. On the other hand, subunit a was not inserted
into membranes that lacked either subunit b or subunit c. The conclusion that subunit a insertion is
dependent upon the co-insertion of subunits b and c differs from the conclusion of several studies, where subunits
were expressed from multicopy plasmids.
H transporting ATP synthases catalyze the
synthesis of ATP during oxidative phosphorylation. Similar enzymes are
found in mitochondria, chloroplasts, and the plasma membrane of
eubacteria (Senior, 1988). The enzymes are composed of two sectors
termed F
and F
. The site of ATP synthesis
resides in the F
sector, which extends from the membrane
surface. The F
sector traverses the membrane and functions
in H
transport. Each sector of the
F
F
complex is composed of multiple subunits in
unusual stoichiometric ratios, i.e.
for F
and a
b
c
for F
in the Escherichia coli enzyme (Foster
and Fillingame, 1982). Each of the three subunits of E. coli F
is thought to traverse the membrane (reviewed in
Fillingame, 1990). The 156-residue subunit b is proposed to
pass through the membrane with a single transmembrane helix near the N
terminus, leaving the bulk of the protein exposed to the cytoplasm.
Subunit a (271 residues) is a polytopic transmembrane protein
with an undetermined number of (perhaps six) transmembrane helices. The
79-residue subunit c folds in the membrane like a hairpin with
its loop exposed to the cytoplasm. The means by which these subunits
are inserted in the membrane and assemble as a complex is unknown. We
report here on the interdependence of subunit insertion into the
membrane, based upon analysis of chromosomal mutant strains defective
in the incorporation of each F
subunit.
A cognate peptide corresponding to residues 2-11 of subunit a, synthesized by the University of Wisconsin Biotechnology Center (Madison, WI), was coupled to keyhole limpet hemocyanin with 2.5% (v/v) glutaraldehyde, using 5 mg of peptide and 10 mg of hemocyanin in 3 ml of 0.14 M NaCl, 0.1 M potassium phosphate, pH 7.5. Following dialysis, the coupled peptide was mixed with either Freund's complete adjuvant for the primary injection or Freund's incomplete adjuvant for secondary injections at 0.17 mg/ml for immunization. A New Zealand White rabbit was immunized with 0.25 ml of emulsified adjuvant at eight sites along the back with booster injections at 2-week intervals. Antibodies against subunit a were detected after 3 months by a peroxidase-coupled immunoassay, using the cognate peptide coupled to bovine serum albumin via 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (Goodfriend et al., 1964) as substrate. The antiserum used in this study was collected during the 17th week of immunization. The antiserum was preabsorbed to LW125 (unc deletion mutant) membranes as described above. Immunostaining was carried out with a 1:1,000 dilution of the antiserum.
Below, we describe mutants of each F subunit,
where the primary mutation abolishes subunit incorporation into the
membrane. The DNA sequence changes were previously described for the uncB mutants (Paule and Fillingame, 1989), for the uncE106 mutant (Mosher et al., 1983), and for the uncF469 mutant (Jans et al., 1985). The ATPase activity of the
mutant membranes are shown in Table 1. The measured activities
are consistent with the previous reports of slightly reduced ATPase
activities in the uncB membranes (Fillingame et al.,
1983; Paule and Fillingame, 1989) and of low ATPase activity in the uncE106 (Mosher et al., 1983) and uncF469 membranes (Jans et al., 1985). The genetic
complementation pattern of the uncE123 and uncF120 mutations were reported by Mosher et al.(1983). The
mutations were subsequently defined by sequencing the respective genes
after amplification by the polymerase chain reaction using the methods
described elsewhere (Fraga et al., 1994a). Representative
immunoblots of the isogenic set of mutant membranes are shown in Fig. 1.
Figure 1:
F subunits incorporated into membranes of mutants bearing mutations
in a single F
subunit. Subunits were detected with
subunit-specific antisera to subunit a (panelA), subunit b (panelB), and
subunit c (panelC) after blotting onto
nitrocellulose paper. The positions of subunit migration and the top
and bottom (tracking dye) of the acrylamide gel are shown. Lane
1, F
standard; lane 2, wild type membrane; lane 3,
uncB
C membrane; lane 4, uncB402 (a W231stop) membrane; lane 5, uncB108 (a W231stop) membrane; lane 6, uncF120 (b R49stop)
membrane; lane 7, uncF469 (b W26stop) membrane; lane 8, uncE123 (c G32R) membrane; lane 9,
uncE106 (c G58D) membrane.
Subunit a was not detected in
any of the mutant membrane samples shown in Fig. 1. Subsequent
experiments with dilutions of wild type membrane indicated that subunit a should have been detected if levels were 20% that of
wild type. In a follow up to these experiments, we tested the limits of
subunit a detection using an antiserum prepared to a cognate
peptide corresponding to residues 2-11 of subunit a.
This antiserum should detect subunit a in mutant membranes at
levels 5-10% of wild type (see Fig. 2). Subunit a was not detected nor was a truncated product observed in any of
the mutant membranes tested, which included uncB402 (a W231stop), uncB108 (a W231stop), uncF120 (b R49stop), and uncE123 (c G23R).
Figure 2:
Subunit a is not detected in
mutant membranes under immunoblotting conditions where the protein
should be detected at levels 5-10% of normal. Subunit a was detected using an antiserum prepared against a cognate peptide
corresponding to residues 2-11 of subunit a. Samples of
wild type membrane (2.5-100 µg of protein) were run in the five left lanes and F standards in the center
lanes. Wild type and mutant membrane samples (50 µg) run in
the six right lanes are as follows: lane 1, wild
type; lane 2,
uncB
C; lane 3, uncB402 (a W231stop); lane 4, uncB108 (a W231stop); lane 5, uncF120 (b R49stop); lane 6, uncE123 (c G32R).
We had previously concluded that subunit a was not
assembled in membranes of uncB402 (a W231amber) and uncB108 (a W231ochre)
mutants (Fillingame et al., 1983; Paule and Fillingame, 1989).
We could infer that subunits b and c were inserted normally
because they were observed in induced membranes of uncB402 and
uncB108 cells (Fillingame et al., 1983;
Mosher et al., 1983) and because the uncB membranes
bound normal amounts of F
(Paule and Fillingame, 1989). The
expected normal incorporation of subunits b and c is
verified here. In the experiments described here and in the study of
Paule and Fillingame (1989), we were unable to detect a truncated
subunit a product. In an independent study, Eya et
al.(1991) did detect truncated subunit a in a series of
chain-termination uncB mutants expressed from plasmids, using
an antiserum that we had generated to an N-terminal cognate peptide
(residues 2-11). In follow-up experiments pursuant to the Eya et al. (1991) report, we were unable to detect the a W231stop truncated product in our own chromosomal strains using
the same N-terminal directed antiserum. The apparent discrepancy may
relate to levels of expression, the background strain, or minor
differences in protocols. (
)
From the experiments
discussed above, we conclude that subunits b and c incorporate into the membrane independently of subunit a.
This conclusion is supported by our analysis of the uncE and uncF mutants, where subunit b or subunit c,
respectively, is incorporated into membranes lacking the other two
F subunits. The independent incorporation of subunits b and c is also observed in chromosomal unc deletion strains expressing the uncF or uncE genes from plasmids (Friedl et al., 1983; Girvin and
Fillingame, 1993). However, as discussed below, the membrane insertion
of a subunit expressed from a multicopy plasmid may not accurately
reflect the normal assembly process from the chromosome.
The second
major conclusion of this study is that subunit a incorporation
into the membrane depends upon a co-incorporation of both subunits b and c. It would be of interest to know whether the
subunits interact as a complex during membrane insertion or whether
subunit a incorporates into a bc complex already in
the membrane. An interaction between subunits b and a during F assembly was suggested previously by Vik and
Simoni(1987). Their analysis of a b G9D mutant showed a lack
of subunit a incorporation into a detergent-extracted
F
Fo preparation. (
)Normal assembly in the b G9D mutant was restored by a P240L subunit a suppressor
mutation. An interaction between subunits c and a during assembly has also been suggested, based upon the analysis
of several point mutations in the conserved polar loop region of
subunit c (Fraga et al., 1994b). In the genetic
background used here (strain AN346), the c R41H mutation
disrupted incorporation of subunit a into the membrane, whilst
the c R41K and c Q42E mutations had no effect on
assembly. In a different genetic background (strain ER) (Felton et
al., 1980), the c R41K mutation reduced membrane
incorporation of subunit a, and the c Q42E mutation
disrupted membrane assembly of both subunit a and subunit b.
The conclusion that incorporation of subunit a into the membrane depends upon a co-incorporation of subunits b and c may be of some surprise. Subunit a is synthesized by in vitro transcription/translation, or
in minicells, from plasmids bearing only the a and c genes (Gunsalus et al., 1982; Klionsky et al.,
1983). In the case of minicells, the amount of subunit a incorporation into a particulate fraction, relative to subunit c, does appear to be significantly reduced on comparing the ac-expressing plasmid to an acb-expressing
plasmid (Klionsky et al., 1983). Further, several laboratories
have shown that overexpression of the subunit a gene from a
plasmid with an inducible promoter can slow the growth of E. coli strains lacking other F
subunits (von Meyenburg et
al., 1985; Eya et al., 1989; Monticello et al.,
1992). Subunit a is thought to be incorporated into the
membrane under these conditions. Conceivably, overexpression could
kinetically force subunit a insertion into the membrane by an
abnormal process. It would be of interest to compare the folding of
subunit a in normal F
with that in membranes where
subunit a was inserted without subunits b and c.