©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Assembly of F Sector of Escherichia coli H ATP Synthase
INTERDEPENDENCE OF SUBUNIT INSERTION INTO THE MEMBRANE (*)

(Received for publication, October 11, 1994 )

Joe Hermolin Robert H. Fillingame (§)

From the Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The F(0) 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(0) 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(0) 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.


INTRODUCTION

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(1) and F(0). The site of ATP synthesis resides in the F(1) sector, which extends from the membrane surface. The F(0) sector traverses the membrane and functions in H transport. Each sector of the F(1)F(0) complex is composed of multiple subunits in unusual stoichiometric ratios, i.e. alpha(3)beta(3)(1)(1)(1) for F(1) and a(1)b(2)c for F(0) in the Escherichia coli enzyme (Foster and Fillingame, 1982). Each of the three subunits of E. coli F(0) 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(0) subunit.


EXPERIMENTAL PROCEDURES

E. coli Strains

The mutant E. coli strains used here are all Ilv transductants of strain AN346 (F, ilvC7, pyrE41, entA403, argH1, rpsL, supE44 (Gibson et al., 1977)). Strain MM180 is the isogenic Ilv/Unc cotransductant. Strain MM188 (uncB402) is described by Fillingame et al. (1983), and strain AN1419 (uncF469) is described by Downie et al.(1981). The other mutant strains are described by Mosher et al.(1983). Strain LW125 bears a chromosomal unc operon deletion in a lon100 derivative of strain AN346 (Ilv, DeltauncBC) (Fillingame et al., 1986). Strain ER (F, asnA31, asnB32, thi-1, relA) is described by Felton et al. (1980).

Preparations and Assays

Cells were grown on M63 minimal medium supplemented with 0.6% glucose, 5 mg/liter thiamine, 0.2 mML-arginine, 0.2 mM uracil, 0.02 mM dihydroxybenzoic acid, and 10% LB (see Miller (1972) for media recipes). Care was taken that cells were harvested in the late exponential phase of growth. Membranes were prepared in TMDG buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl(2), 1 mM dithiothreitol, 10% (v/v) glycerol) by rupture of cells in a French press (Mosher et al., 1983). F(0) was purified according to Schneider and Altendorf(1984). ATPase activity was assayed at 30 °C with 0.4 mM [-P]ATP (Fillingame, 1975). Membrane protein concentrations were determined by a modified Lowry method, after solubilization with SDS, using bovine serum albumin as a standard (Fillingame, 1975).

Antisera

Rabbit antisera specific to subunit a (Deckers-Hebestreit and Altendorf, 1986), subunit b (Perlin and Senior, 1985), and subunit c (Girvin et al., 1989) were diluted 1:750, 1:1,000, and 1:1,000, respectively. Prior to dilution, the anti-subunit sera were preabsorbed with membranes prepared from unc operon deletion mutant (strain LW125) or a strain lacking subunit b to remove antibodies that nonspecifically cross-reacted with E. coli membrane proteins (see Perlin and Senior(1985) and Girvin, et al.(1989)).

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.

Immunoblotting

Membrane vesicles were incubated in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 2.5% beta-mercaptoethanol, 10% (v/v) glycerol, and 0.01% bromphenol blue) at 5 mg of protein/ml at room temperature for 1 h. Heat treatment was avoided as this led to significant aggregation of subunit c. The solubilized membrane proteins (50 µg/lane) were electrophoresed on a 15% polyacrylamide slab gel (15% acrylamide, 0.25% bisacrylamide, containing 0.4% SDS) using the Tris-glycine electrophoresis buffer described by Laemmli(1970). Proteins in the gel were transferred electrophoretically onto nitrocellulose paper and blocked essentially as described by Towbin et al.(1979), with a blocking mixture usually composed of 1% gelatin and 2% bovine serum albumin. Immunoblotting with the antiserum to the subunit a cognate peptide was carried out after blocking with a suspension of 5% nonfat powdered milk (Harlow and Lane, 1988). Immunostaining was carried out using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA).


RESULTS

Below, we describe mutants of each F(0) 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(0) subunits incorporated into membranes of mutants bearing mutations in a single F(0) 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(0) standard; lane 2, wild type membrane; lane 3, DeltauncBC 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.



Subunits b and c Incorporate Independently of Subunit a

Subunit a was not detected in membranes of two a W231stop mutants. We were also unable to detect truncated products of subunit a. Normal amounts of subunits b and c were observed in membranes of both mutants. Several bands show minor differences in the staining intensity, but consistent differences between bands were not seen in several independent blots of the same membrane fractions.

Subunits b and c Are Required for Membrane Insertion of Subunit a

Subunit b was not detected in membranes of two chain termination uncF mutants (i.e.b W26stop and b R49stop). Normal amounts of subunits c were seen in both membranes. In contrast, subunit a was not detected in either of the uncF membrane fractions. Subunit c was not detected in membranes of two uncE point mutants, i.e.c G32R and c G58D. Normal amounts of subunit b were seen in the membrane of both mutants. In contrast, subunit a was not seen in either uncE membrane fraction. Hence, subunits b and c incorporate independently into membranes lacking both of the other two F(0) subunits. Incorporation of subunit a, on the other hand, requires the presence of both subunits b and c.

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 geq20% 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(0) 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, DeltauncBC; lane 3, uncB402 (a W231stop); lane 4, uncB108 (a W231stop); lane 5, uncF120 (b R49stop); lane 6, uncE123 (c G32R).




DISCUSSION

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(1) (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. (^1)

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(0) 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(0) 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(1)Fo preparation. (^2)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(0) 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(0) with that in membranes where subunit a was inserted without subunits b and c.


FOOTNOTES

*
This study was supported by U. S. Public Health Service Grant GM23105 from the National Institutes of Health and by a grant from the Human Frontiers Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 587 Medical Sciences Bldg., University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-1439; Fax: 608-262-5253.

(^1)
Using uncB402 DNA, a product of the approximate size expected for the truncated subunit was synthesized in an in vitro transcription-translation system, and a possibly corresponding band was observed in membranes of uncB402 cells following unc overexpression (Fillingame et al., 1983). On subsequent Western blotting of the induced uncB402 membranes with the antiserum to the N terminus of subunit a (cognate peptide corresponding to residues 2-11), we did not detect a truncated product. We conclude that the truncated protein is synthesized but then degraded relatively rapidly.

(^2)
In an earlier study, Friedl et al. (1981) concluded that subunits a and c were both incorporated into membranes of a chromosomal mutant lacking subunit b, but the antiserum used to detect subunit a was later found to be directed against a contaminant in the F(1)F(0) preparation used as antigen (Friedl et al., 1983).


ACKNOWLEDGEMENTS

We thank Drs. Gabriel Deckers-Hebestreit and Karlheinz Altendorf (University of Osnabruck) and Drs. David Perlin and Alan Senior (University of Rochester) for the gifts of antisera to subunits a and b, respectively.


REFERENCES

  1. Deckers-Hebestreit, G., and Altendorf, K. (1986) Eur. J. Biochem. 161, 225-231 [Abstract]
  2. Downie, J. A., Cox, G. B., Langman, L., Ash, G., Becker, M., and Gibson, F. (1981) J. Bacteriol. 145, 200-210 [Medline] [Order article via Infotrieve]
  3. Eya, S., Maeda, M., Tomochika, K.-I., Kanemasa, Y., and Futai, M. (1989) J. Bacteriol. 171, 6853-6858 [Medline] [Order article via Infotrieve]
  4. Eya, S., Maeda, M., and Futai, M. (1991) Arch. Biochem. Biophys. 284, 71-77 [Medline] [Order article via Infotrieve]
  5. Felton, J. S., Michaelis, S., and Wright, A. (1980) J. Bacteriol. 142, 221-228 [Medline] [Order article via Infotrieve]
  6. Fillingame, R. H. (1975) J. Bacteriol. 124, 870-883 [Medline] [Order article via Infotrieve]
  7. Fillingame, R. H. (1990) in The Bacteria (Krulwich, T. A., ed) Vol. 12, pp. 345-391, Academic Press, Orlando, FL
  8. Fillingame, R. H., Mosher, M. E., Negrin, R. S., and Peters, L. K. (1983) J. Biol. Chem. 258, 604-609 [Abstract/Free Full Text]
  9. Fillingame, R. H., Porter, B., Hermolin, J., and White, L. K. (1986) J. Bacteriol. 165, 244-251 [Medline] [Order article via Infotrieve]
  10. Foster, D. L., and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009-2015 [Abstract/Free Full Text]
  11. Fraga, D., Hermolin, J., and Fillingame, R. H. (1994a) J. Biol. Chem. 269, 2562-2567 [Abstract/Free Full Text]
  12. Fraga, D., Hermolin, J., Oldenburg, M., Miller, M. J., and Fillingame, R. H. (1994b) J. Biol. Chem. 269, 7532-7537 [Abstract/Free Full Text]
  13. Friedl, P., Bienhaus, G., Hoppe, J., and Schairer, H. U. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 6643-6646 [Abstract]
  14. Friedl, P., Hoppe, J., Gunsalus, R. P., Michelson, O., von Meyenburg, K., and Schairer, H. U. (1983) EMBO J. 2, 99-103 [Medline] [Order article via Infotrieve]
  15. Gibson, F., Cox, G. B., Downie, J. A., and Radik, J. (1977) Biochem. J. 162, 665-670 [Medline] [Order article via Infotrieve]
  16. Girvin, M. E., and Fillingame, R. H. (1993) Biochemistry 32, 12167-12177 [Medline] [Order article via Infotrieve]
  17. Girvin, M. E., Hermolin, J., Pottorf, R., and Fillingame, R. H. (1989) Biochemistry 28, 4340-4343 [Medline] [Order article via Infotrieve]
  18. Goodfriend, T. L., Levine, L., and Fasman, G. D. (1964) Science 144, 1344-1346
  19. Gunsalus, R. P., Brusilow, W. S. A., and Simoni, R. D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 320-324 [Abstract]
  20. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual , pp. 471-509, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  21. Jans, D. A., Hatch, L., Fimmel, A. L., Gibson, F., and Cox, G. B. (1985) J. Bacteriol. 160, 764-770
  22. Klionsky, D. J., Brusilow, W. S. A., and Simoni, R. D. (1983) J. Biol. Chem. 258, 10136-10143 [Abstract/Free Full Text]
  23. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  24. Miller, J. H. (1972) Experiments in Molecular Genetics , pp. 431-435, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  25. Monticello, R. A., Angov, E., and Brusilow, W. S. A. (1992) J. Bacteriol. 174, 3370-3376 [Abstract]
  26. Mosher, M. E., Peters, L. K., and Fillingame, R. H. (1983) J. Bacteriol. 156, 1078-1092 [Medline] [Order article via Infotrieve]
  27. Paule, C. R., and Fillingame, R. H. (1989) Arch. Biochem. Biophys. 274, 270-284 [Medline] [Order article via Infotrieve]
  28. Perlin, D. S., and Senior, A. E. (1985) Arch. Biochem. Biophys. 236, 603-611 [Medline] [Order article via Infotrieve]
  29. Schneider, E., and Altendorf, K. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7279-7283 [Abstract]
  30. Senior, A. E. (1988) Physiol. Rev. 68, 177-231 [Free Full Text]
  31. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  32. Vik, S. B., and Simoni, R. D. (1987) J. Biol. Chem. 262, 8340-8346 [Abstract/Free Full Text]
  33. von Meyenburg, K., Jorgensen, B. B., Michelsen, O., Sorensen, L., and McCarthy, J. E. G. (1985) EMBO J. 4, 2357-2363 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.