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Elsevier
BBA 86106
Contents
Introduction
III Topographical studies ... 0 •• 0 ••• • 0 0 ••••••• 0 0 • • ••••••••• 0 ••••••••• • 0 •••• 0 0 •• 0 0 0 0 0 0 0 0 0 • 0 0 0 •••• 0
4
A. Studies with proteinases .. . ... . . . ...... . 0 0 0 •• 0 •••••• •• 0 •• • • 0 • 0 •••••••••••• 0 0 0 0 0 0 •••••••• •••
4
Bo Studies with antibodies .. 0 0 • 0 •• • •••• •• ••••• •• • •••• 0 0 0 0 0 • 0 • 0 • 0 •• 0 •• •• • 0 • • 0 0 0 0 0 0 0 ••••••• • ••• 6
C. Surface label 0 •• 0 ••••••••• 0 • • 0 •••• •• •••••••••••• •• 0 • • • •••••••••••••• •• • ••• 0 0 •• 0 • 0 0 •• 0 0 0 0 6
D. Cross- linking experiments .... . ... .. . 0 •••••••••• • •••• 0 0 ••••••••••••••• • •••• 0 0 0 •• 0 • • 0 0 •••••• 9
E. Circu lar dichroism measurements . ... . 0 0 0 0 0 • 0 0 •• •••• 0 0 • 0 0 • 0 • •• •• •• 0 • 0 0 0 0 0 0 0 0 0 •• 0 • 0 ••••••••••• 10
Referen ces . . . ....... . .. . ...... ... ...... . ... .. .... . . .. ............. . .... . . .............. ..... 24
I. Introduction
Abbreviations: DCCD, N,N'-dicyclohexylcarbodiimide; PTH,
phenylthiohydan toin; T ID, 3- trifluormethyl-m-(iodophenyl)-
diazirine; NCCD, N-(2 ,2,6,6-te trame th ylpiperidyl-l-oxyl)- T he membrane-integrated part Fo of the ATP
N'(cyclohexy l)carbod iimide. sy nthase translocates protons across the mem-
mediated proton transport is tightly coupled to the E.\'cheriehia eoli. The ATP synthase of this organism
synthesis or hydrolysis of ATP catalysed by the can now be prepared in large quantities with a
aß l'
F I -A TPase part. By the removal of FI , the proton purity of at least 90% due to the work of Foster
pathway in Fo is opened, and can be studied
independently. Fo represents, therefore, an interest-
F, Fa I I I and Fillingame [7] and Friedl et al. [8]. From these
preparations the Fo part has been isolated by 1 O~ 110 1I ~ 170
IIp-l\la_l'ro_!.I'u_l\la_Ll'll_Thr_IJe_f'he_\'Hl_lrp_Val_!'!ll'_ll'u_!\cl_I\[;Il_I"u_II,'I_/\,;p_leu_
various procedures [9-11]. It contains three sub-
ing and up to now unique system for analysis of (i)
the structure and function of a proton conductor,
Fa I I units a, band c (X, l/; and w, or proteolipid or 11,;' I 'j(J I ~~ 1 MI
Q b DCCD-binding protein) as shown in Fig. 1. The
\' u I-Va 1-1' ro -S{' r-'\ 1 (l ~'\'l!'- \llll_II!,n_Va 1- I 1>1 -I. ('u-S" r~ll" l~'\ 1 11_1 eu_G I y _I' al-!'I1<' _! 1 <'_I eu-
and (ii) the coupling of the Fo-mediated proton
transport to the hydrolysis or synthesis of ATP whole ATP synthase contains, in addition, the five
occurring in the FI -ATPase. F I subunits IX, ß, y, 8 and E. These eight subunit
Several recent reviews covered the genetics of polypeptides are coded for by the eight genes that
Genes (atp aperan)
the ATP synthase [1,2], the nucleotide sequence of form the alp (une) operon (see section IV). The alp
_._------_.- operon has been weIl defined geneticaIly, espe- 211'> ) ',0 2)~ <'60
the ATP synthase genes of E. eoli (Walker, J.E., ! rp-'\ I il-l J,' _1'1>,,_11 J H-Il <'_I. pu_I 1 {'~ 11 {'~ Th 1'_1 pu_G 111_'\ 1 a-!'IH'~ I 1 {'_I'lle_llp I ~ \.' "J-l ,'u-l h I _
Saraste, M. and Gay, NJ., unpublished results), Q C b a l' ß cially by Downie, et al. [1,12], and by Von Meyen-
the proton-A TPase from bacteria and mitochon- burg and co-workers [13], and its nucleotide se- , I) 10
1-lkl_'\"II_1 ('u_,\"n_Alo_lhr_1 1,>··,\I,,_I'I\\'_lnl_\ (>u-I'I1,>_\';ll_1 <'u-!'I",_
271 79 156 177 513 287 460 133 quence has been determined [14-23]. Therefore,
dria [4], the structural basis of proton-translocat-
ing protein function [5], and the structure and the subunit composition of the ATP synthase and
genetics of the proteolipid subunit of the ATP Fig. 1. Subunits and genes of the E. coli ATP-synthase. The Fo from E. eoli is proven. The amount of protein of
synthase [6]. This review tries to collect new pro- ATP synthase F1Fo and the Fo were isolated from E. coU R) subunits a, band e corresponds to molar ratios
teinchemical data on Fo and to combine them with according to Friedl et al. [8,10] and analyzed by SDSjpoly- of 1: 2 : 10 according to Foster and Fillingame
acrylamide gel eleetrophoresis. The order of the genes in the
genetical and functional data in order to give the [24], or 1 : 2: 12 according to Von Meyenburg and
une operon and the eorrelation of genes with A TP-synthase
present-day knowledge on the structure-function subunits was established genetieally and by nucleotide se-
co-workers [25]. For the mitochondrial [26] and
relationships in this proton conductor. Although quenee analysis [1,12-23]. The numbers below the genes indi- chloroplast [27] A TP synthase, six proteolipid
detailed and definite information has been ob- eate the number of amino acid residues in the subunit poly- (subunit e) molecules have been determined. These
peptide. different stoichiometries are difficult to explain for
tained on several structural and functional aspects
of Fo, we are still far from an understanding of the the time being. lt is quite clear, however, that
proton transport at a molecular level. subunit c exists at least in six copies per R). The
ingly, our present knowledge on the proton-con- dimeric form of subunit b is also indicated by
11. Background, problems, and approaches cross-linking experiments (see subsection lUD). Fig. 2. Amino acid sequenees of the Fo-subunits a, b, and e of
ducting Fo part is derived from studies of the
The iso la ted Fo from E. eoli can be reinserted the ATP synthase from E. eoli.
The ATP synthase occurs in remarkably similar bacterial, mitochondrial or chloroplast protein,
form in procaryotic and eucaryotic cells. Accord- each offering special experimental possibilities. In into artificial membranes, and proton conductance
can be measured in response to a valinomycin-in-
duced potassium diffusion potential [9-11] (Table genes. The derived amino acid sequences of the
TABLE I I). The proton conductance is inhibited strongly three Fo subunits are presented in Fig. 2. They
by DCCD and by added FI . Reconstituted Fo have been extensively evaluated by means of com-
ACTIVITIES OF F)f'o FROM ESCHERICHIA COLI
rebinds FI , and, thereby, ATP-dependent proton puter programs with respect to secondary structure
Values are taken from Ref. 74. predictions, polarity profiles and homologies with
translocation is restored. The data obtained with
isolated Fo are compiled in Table I together with corresponding subunits from other organisms
Fraction Electro-impelled 1'1'" conduetion ATP-dependent
H'" translocation corresponding data measured with Fo in whole [3,4,28,29]. A summary of such evaluation is also
p H -electrode Fluorescence test
Fl uorescence test membranes. Accordingly, both proton conduc- shown in Figs. 12, 13 and 15 in the last section of
(nmol· min - I . mg")) (UF1·mg- 1)
tance and functional rebinding of FI can be used this review.
Membranes 130 to analyse Fo. The established subunit composition and cova-
F)-depleted
The nucleotide sequence of the genes of all lent structure of the E. eoli Fo reflect a major
membranes 44 65
Liposomes with three F~ subunits of E. eoli was determined inde- achievement in the characterization of the A TP
F)I"o 5 16 2330 pendently by three groups in 1981 [15,17,21]. synthase proton channel. However, for the eluci-
Fo +F) 41 151 1910 Amino~terminal amino acid sequences of the iso- dation of Fo-mediated proton conductance, addi-
Fo 870 2565 1 lated subunits a, b, and e were established [17] in tional information is needed (i) on the three-di-
Fo + DCCD 2 24
order to prove the start of the individual subunit mensional structure of the protein, and (ii) on the
4 5
funetional amino aeid residues and domains eon- Several approaehes were applied to identify the observed, whieh remained firmly bound to the
stituting the proton pathway and partieipating in funetional amino aeid residues and domains in the membrane, as treatment with high salt, urea or
the eoupling of Fo and F 1 funetions. Fo. Due to the ubiquitous oeeurrenee of Fo, it is guanidine hydroehloride failed to remove this frag-
A high resolution of the three-dimensional feasible to eompare the amino aeid sequenees of Fo
(Y-
[3- ment from the membrane. By proteinehemieal
strueture of Fo will be aehieved finally only by subunits from very distantly related organisms. analyses it was shown that ehymotrypsin did not
X-ray analysis after erystallization of the protein. Based on the assumption that the meehanism of ,- cleave at the primary cleavage sites phenylalanine,
With other integral membrane proteins, two-di- proton eonduetanee remained unehanged during Q -
tyrosine, tryptophan or methionine, whieh are all
o- loeated in the hydrophobie N-terminal segment of
mensional erystals [30-34] and, more reeently, also evolution, it ean be expeeted that the group of b-
three-dimensional erystals [35-37] have been pro- invariant amino aeids also eomprises the fune- the subunit b. Thus, cleavage had oeeurred about
(-
dueed. Crystals of the Fo protein have not yet been ti on al residues. 25 residues away from the C-terminus.
obtained. Anyway, X-ray analysis of eomplex pro- Chemieal modifieation may lead to the iden ti- (-
Under eonditions where only subunit b was
teins, as Fo, will be exeeedingly eomplieated, and fieation of funetional residues, provided that the degraded by proteinases (see below), eleetro-
will probably always depend on auxiliary informa- reaetion of a defined residue ean. be eorrelated impelled H +-eonduetion (passive proton permea-
A B 1 2 3 4 5 6 7 8 9 10 11 12 bility) was not affeeted. Similar results were re-
tion from other struetural studies. with an inhibition of Fa funetions.
As deseribed in subseetions IIlA-C, the surfaee Moleeular geneties and genetie engineering offer ported by Sone et a!. [48] after degradation of the
Fig. 3. Proteolytic treatment of Fo subunits in Fj-depleted
of Fo has been mapped by probes whieh have further promising possibilities to study funetional 13 kDa subunit of the PS-3 Fo. But in contrast to
membranes. Fj-depleted membranes of E. coli were prepared
aeeess either only from the polar water phase or properties of Fo. Espeeially in E. coli, but also in from an ATP synthase-overproducing strain [49]. Lane A shows PS-3, in E. coli F1 eould be still bound to pro-
from the hydrophobie lipid phase. Proteases and yeast, a variety of mutants whieh eontain a defee- separated Fjl'O-, and lane B, separated Fj-depleted membranes. teinase-treated membrane. The rebound ATPase
antibodies are valuable tools, not only for identifi- tive Fo have been isolated. In some of these strains, The proteinase concentrations, increase in the sampIes from left aetivity was not sensitive towards DCCD and
to right for V8 Clanes 1-3), chymotrypsin [4-6], trypsin [7-9] ATP-dependent proton transloeating was not re-
eation of polar domains but also for the de- Fo appears to be normally assembled, and, there- and subtilisin [10-12]. The star indicates the digestion product
termination of the orientation of the polypeptide fore, funetional residues might have been modified eonstituted. These results indieate that the polar
obtained with chymotrypsin. The arrow indicates the possible
ehains in the membrane. The surfaee of Fa aeeessi- by the mutation. The altered residues might be cleavage product from subunit a. part of subunit b plays an essential role in eou-
ble from the lipid bilayer has been studied with identified by protein or DNA sequenee analysis. pling the funetions of F1 with the H+ transloeation
photoreaetive membrane-permeating probes. Espe- Furthermore, the funetion of the different Fo sub- aeross the membrane. I t is a distinet possibility
eially, earbene labels of broad speeifieity offer units has been studied by eonstrueting strains that the proteinase-sensitive eomponent at the in-
promising possibilities, sinee they should aet like a where one or two of the Fo subunits are missing. terphase of Fa and F 1 from rat liver [50] also
razor blade and modify all aeeessible residues at eorresponds to the E. coli subunit b.
the protein/lipid interphase. Not only would this I1I.Topographical studies teinases. F1 binding and ATP-dependen t H + trans- Subunit a was not degraded by ehymotrypsin
allow an identifieation of membrane-embedded loeation were lost after digestion, indicating a and trypsin, while proteinase V8 and subtilisin at
polypeptide segments, but, in addition, the pattern JIJA. Studies with proteinases binding aetivity of the extramembranous part of high eoneentrations were effeetive (Fig. 3). Sub-
of aeeessible residues might yield information on the 13.5 kDa protein. Corresponding studies on tilisin, at lower eoneentrations, digested all mem-
seeondary struetures of the polypeptide ehain. The assymmetry of membrane proteins has been the proteinase sensitivity of the Fo-subunits from brane proteins exeept subunit a to produets with a
The study of subunit eontaets in Fo by eross-lin- investigated in many instanees by proteinase treat- E. coli [49] gave similar results. Experiments were M, of less than 15000. The appearanee of the
king experiments is handieapped (i) by the low ment of whole membranes and analysis of the earried out using F1-depleted everted membrane polypeptide with a M r of 22000 at high er subtilisin
eontent of reaetive side-ehains and (ii) by the cleavage produet [38--41]. The sidedness of the C- vesicles from strain CM 2786, whieh overproduees eoneentrations was thus indieative for a cleavage
eomplex subunit eomposition. These diffieulties and N-terminal segments as weil as the loeation of ATP synthase about 5-fold (see also subseetions produet of subunit a. Possibly, the quite polar
eould be overeome partially by using photo- some internal polar loops have been determined, IV A and B). Thus, the three Fa-subunits eould be N-terminal segment was attaeked by the pro-
aetivated bifunetional reagents for eross-linking for example, in the baeteriorhodopsin, by treat- easily identified in wh oie membranes by SDS- teinase and, thus, might be loeated at the eyto-
and speeifie antibodies for the identifieation of ment of the purpIe membrane with papain, trypsin polyaerylamide gel eleetrophoresis in eombination plasmie side of the membrane.
eross-linked subunits. and ehymotrypsin [42-44]. These studies, together with a silver stain method (Fig. 3). Subunit b was Subunit c was not or only slightly affeeted by
Further information on the strueture of Fo may with erystallographie data [30,31] and the primary most sensitive towards proteinase treatment. any of the proteinases. Even prolonged ineubation
be derived from the analysis of Fo mutants. Amino strueture [43,45], provided information on the Trypsin, subtilisin and proteinase V8 produeed times and high eoneentrations of proteinase did
aeid residues altered in DCCD- or oligomyein-re- folding and orientation of this polypeptide in the small fragments, indieating that large portions of not lead to any extensive degradation (ineubation
sistant mutants might be involved in the binding membrane [46,47]. the pro tein were aeeessible to proteinases. These time 5 days, at a ratio of proteinase to membrane
of the inhibitors, and might therefore be loeated in First experiments on proteinase treatment of proteinases digested subunit b eompletely, demon- protein of 1: 5) [49]. Sinee subunit c, and espe-
proximity to eaeh other at the surfaee of the isolated Fa from PS-3 were deseribed by Sone et a!. strating that the polar domain of subunit b extends eially the polar domain, is very resistant to pro-
moleeule. Subunits from assembly-defieient mu- [48]. An Fa subunit of M, 13 000 was readily to the eytoplasm in E. coli, sinee everted mem- teolysis even in the denatured state, this result
tants might be alte red in residues involved in digested with trypsin and nagarse, but the proton brane vesicles were used. With ehymotrypsin, how- does not neeessarily imply that the funetional pro-
intermoleeular or intramoleeular protein eontaets. permeability of the Fn was not affeeted by pro- ever, a defined cleavage produet of M r 15000 was tein is eompletely inaeeessible to proteinases.
6 7
NO,
- 'Y
a
o
a-
loo
~~.--.
I
10
-""",
JO
10
1r
20 30 40 50
Ii
00 70
I sible from the lipid phase.
After reaction of [ 125 I]TID with a functional Fo
(FI-depleted or F1Fo), only a few residues of sub-
R.. ldue
problems with this type of precursor have been unit c were attacked (Fig. 8A-B) in contrast to the
B
discussed recently [70]. Cpm multiple labelling in the N-terminal segment of
B
To circumvent this problem, trifluoromethyldi- cpm subunit b. Labelling is restricted to four to six
azirines have been introduced by Brunner [68]. 300 residues in the N-terminal segment and to five to
The undesired diazoisomer is stabilized by the six residues in the C-terminal segment. No label-
strong electron-withdrawing trifluoromethyl group, 10000 ling occurred in the middle of the polypeptide
and esterification by the diazoisomer is thus 200 chain from positions 20-52, despite the fact that
M
negligible. Fig. 5 C and D shows that all three Fo the sequence from residues 20-33 is highly
subunits in the isolated complex FI Fo are labelIed enriched in hydrophobic residues, and thus is a
100
by the carbene, roughly according to their protein F
possible candidate for a membrane-embedded seg-
mass. In FI-depleted membran es, the same label- ment. The labelling pattern changes dramatically
ling pattern of subunits a and b is observed, the --,
M Y
if [ 125 I]TID is reacted with the SDS-solubilized
10 20 30 F
labelling of subunit c is obscured by the large isolated subunit c (Fig. 8C). LabelIed residues are
Y
amount of labelIed phospholipids. distributed over the whole length of the poly-
Labelling patterns were recorded with (i) iso- cpm
C Ll__ t
10
'I ~
20 30 40 50
I 50 70
peptide, including the hydrophobic segment 20-30,
lated ATP synthase (FI Fo ), (ii) F I depleted mem- Auidue
which was not labelIed in the functional subunit c.
branes from strain CM 2786 which overproduces Apparently, these residues are shielded by other
500 -----
the ATP synthase, and (iii) subunits denatured in ... protein segments in functionally assembled sub-
a 2% SDS-solution. cpm
C unit c.
e25 I]TID labelIed subunits band c could be
10000 In the functional subunit c, the [125 I]TID mod-
isolated and the modified residues could be de- ified residue at the N- and C-terminals exhibit a
termined. Sequence analysis was performed by 100 \ remarkable repeat. They occur at positions 4, 8,
automated solid phase Edman degradation. Re- 10--~-20-----~ 10, 11, 15, 19 and 53, 57, 65, 73, 76 [74]. Such
leased PTH amino acids were identified by thin- ... n + 3 or n + 4 patterns are typical for residues
Fig. 7. Identification of amino acids of subunit b, labelIed with ... y
layer chromatography and associated 125I-radioac- [ 125 I]TIO (Hoppe, J., Bronner, J. and J~rgensen, B.B., unpub- y K
located on the same side of an a-helix.
tivity was measured as shown in Fig. 7A-C. The lished data). After labelling, subunit b was isolated from F 1Fo F MG
reaction product with e 25 I]TID was further by HPLC gel permeation chromatography. For F 1-depleted
...
F G
G
J II D. Cross-linking experiments
analyzed by autoradiography of the thin-layer
plates. This allowed the identification of labelIed
membranes, proteins were separated on aSOS poly acryl amide
gel and the band corresponding to subunit b was eluted from
11 r i ;~;~i I II I The subunit contacts in the E. coU Fo were
the gel strip. Subunit b from (A) F 1Fa in Aminoxid WS35
20 30 40
amino acid residues even over a high background buffer, (B) F 1-depleted membranes, (C) F 1Fa in SOS-buffer ...
10 50 60 70
studied with the isolated complex FI Fo by cross-
and an overlap from the preceding amino acid Arrows indicate labelIed amino acid residues, identified by Fig. 8. Labelling of individual amino acids of subunit c by linking experiments. Fluoronitroazidophenyl was
residue. autoradiography of the PTH amino acids after thin-layer chro- C25 I)TIO (Hoppe, J., Brunner, J. and J~rgensen, RB., unpub- used, which in the first step binds by a nucleophilic
matography.
Proteinchemical analyses of TID-labelled sub- lished data). Labelling was determined by sequencing the whole displacement reaction to the side-ch;üns of cy-
protein, attached to gl ass beads. Sampies wcre analyzed for steine, lysine, tyrosine or histidine. Subsequently,
unit b by CNBr cleavage showed that more than
125I-radioactivity and labellcd amino acids wcrc dctectcd by
98% of the label was attached to the N-terminal au(oradiography of thc released PTH amino acids after thin-
segment. Fig. 7A, B depict the labelling of individ- membranes. In the N-terminus of subunit b, again layer chromatography. Thc amount of label bound to an amino
e
ual amino acid residues after reaction of 25 I)TID Cys-21 received the bulk label. But other amino acid side-chain was then calculated, assuming a repetitive yield from F 1Fa in Aminoxid WS 35 buffer; (B) subunit c in F 1-de-
with the isolated complex FI Fo- or FI-depleted acids are significantly labelIed. Most interestingly, of 95% for each cycle of Edman degradation. (A) Subunit c pleted membranes; (C) subunit c in an SOS solution.
10 11
~
lCJO )'>(':1 n~"
...J_,_.-,.........>-.-,-.. ~."_,._. __ •.. -,_j_.-L-_ ,
I,()'J
scribed, where both the genotypes and the pheno- TABLE III rebind F] and to conduct H ' [102] (Table III).
:::::::==::::::=.::::::=:::=::: [:::::::::::::'::;:::::=-.::1 C::::;;:::::::J c::=::::::..:=-~.~---'._'=
typic alterations have been studied in so me detail. ACTIVITIES OF F'o IN unc-MUTANTS LACKING INDI- Also included in these studies was a mutant mem-
b b ,
VIDUAL Fo SUBUNITS [102] brane which is devoid of subunit e due to a point
pRPG ~5 1 VB. Deletion mutants and plasmids Strains: A 1 wild-type; CM 1470 and CM 2080 deletion mutation in the alp E (une E) gene (see below).
pf?P[) 51
pRPG SB
rnutants; the presence of subunit b depends on growth phase The results are summarized in Table IH. The
The deletion mutants of E.coli, compiled in Fig. (log = logarithmic, stat = stationary phase); AM 12 and DG membranes of these strains comprise all possible
pOM lH
10/6, polar mutants; pOM 11-1, plasmid with structural gene
10, allow the study of membranes in wh ich selected for subunit a; pRPG 51, plasmid with structural gene for
subsets of Fa subunits. None of the Fa subunits
(1'12080
Fa subunits are missing. Strain CM 2080, char- subunits band S [l02]. alone and none of the combinations of two Fa
(M 11,70
acterized in some detail, has adeletion of about subunits allows a H + conductance across the
Fig. 10. Plasmids expressing selected genes of the alp operon in 500 basepairs at the start of the subunit a gene Strain Fo H+ con- F! membrane. Subunit a, as well as b, each present
E. eoli and strains carrying defined deletion in the alp operon. subunits duction binding
c=J, coding region for the subunits a, e, b; - - , indi- [102]. Membranes of this deletion mutant contain individually in the membrane, promote the bind-
cates the cloned region of the alp operon in the respective Fa subunits band e, but only if isolated from a b c + +
ing of F], while subunit e alone does not. Accord-
A 1
plasmid [l01,102]; ------, indicates the deleted region in the exponentially growing cells. If membran es are pre- DG 10/6 a b + ingly, each pairwise combination of two Fa sub-
two strains [13]. pared from mutant cells in the stationary growth CM 2080 (log) b c + units allowed arebinding of F] to the membrane.
phase, only subunit e is detected. Most likely, AM 12 a c + The observed physical bin ding of F] to incomplete
subunit b was proteolytically degraded in sta- CM 1470+pOM 11-1 a + Fa must be different from the binding to intact Fa.
CM 1470 + pRPG 51 b +
tionary phase cells [49]. As described in subseetion c
The rebound F] -ATPase is active, although the
CM 2080 (stat)
genes encoding subunit a and c are covered by lIlA, the large polar domain of subunit b is easily CM 1470 proton conductance is abolished in the incomplete
mutations leading to defective ATP synthase (Pho attacked by proteinases in vitro [49]. Another dele- Fa·
I, Pho 11), or resistance towards inhibitors (Oli I, tion mutant CM 1470 has a large deletion includ-
IVC. Point mutations
Oli 11, Oli III, Oli IV) [93-·96]. The corresponding ing genes of subunits a, C, b, 0 and part of a [13]. The membranes of all these strains, in which
nuclear subunit c gene in N. crassa can also be The membrane of this strain is devoid of all Fa single or multiple Fa subunits are genetically de- IVC-l. Mutants with defective Fo
mutated to an allele leading to oligomycin resis- subunits. leted, have been analysed for their capability to All mutant strains compiled in Table IV revert
tance [97]. In other types of mutant, a Mu phage has been
In E. coti, phages and plasmids have been iso- inserted at various points of the atp operon. The TABLEIV
lated containing all FaF] subunit genes, i.e., the inserted phage DNA prevents the efficient tran- E. COLI MUTANT STRAINS WITH DEFECTIVE OR DCCD-RESISTANT F o
whole atp operon [98-101]. This allows, for exam- scription of all genes located downstream. These n.d., not determined.
pIe, the construction of strains where the level of polar mutations, therefore, result in membranes
FaF] in the membrane is increased several-fold which are devoid of certain subunits. One example Genotype Affected Amino acid Complemen- Presence in H + conduc- ATPase acti- Ref.
[48]. The membranes of such overproducing strains is the strain AS 12 (Table IV) [103,104], where the or strain subunit exchange tation: the membrane ti on vity
dominante + ),
offer advantages for structural studies. Further- Mu phage is inserted in the atp A gene coding for
recessive( )
more, plasmids are now available containing only F] subunit a. Accordingly, F] subunits a, y, ß, and
DG 25/3 a n.d. n.d. + 105
segments of the atp operon [101,102]. Thus, a ( are not expressed. In addition, Fa subunit b is not
subset of subunits or in certain cases even a single present in the membrane. Possibly, the defect in F] DG 25/9 b n.d. + + + 105
prevents either the assembly of subunit b or facili- une F 469 b n.d. 12
subunit can be expressed at an increased level in
the cell (Fig. 10). The latter plasmids are useful, tates a proteolytic degradation. DG 7/1 ASP6!-Gly + + + 108
especially when introduced into mutant strains Finally, several segments of the atp operon have DG 18/3 e ASP6!-Asn + + + 109
been cloned on plasmids. One plasmid, pOM 11, DG 27/10 c Ala 2 !-Val + + (Hoppe, 1.,
where large parts of the atp operon are deleted or
contains the intact coding sequence for subunit a Schairer, H.U.
not expressed. and Von Meyen-
In principle, a mutation in a subunit gene can [13]. A functional subunit a is expressed from the burg, K.
affect the properties and function of Fa at several cloned gene, since it complements the deletion of
une E 463 e Leu)! -Phe _( +) a _( +) a -(+)"- 2, 111
levels. (i) The mutation may prevent the bio- the subunit a gene in strain CM 2080. Another ( ulle E 408)
synthesis of a subunit or its integration into the plasmid, pRPG 51 [101], contains the sequences
membrane. (ii) A mutation may affect the assem- coding for subunits band o. The cloned genes are ulle E 410 c Pro64 -Leu + + + 12, 2.
bly of Fa, even if the mutated subunit is integrated expressed. The plasmids have been introduced into UIlC E 429 C GlY2)-Asp 12,111
into the membrane. (iii) A mutation may affect the the deletion mutant CM 1470, where all Fa genes DC 13 c Ile 28 - Val n.d. + + + 115
function of Fa' even if the altered subunit is assem- are missing [13]. The strains obtained contain in DC25 c Ile 28 - Thr n.d. + + + 115
bled into Fa. the membrane either only Fa subunit a or only
In the following, mutant strains will be de- subunit b. a At high gene dosage.
14 15
readily, and consequently originated from single tive, whereas in mutant DG 25/9 subunit b is with apparently assembled Fo, the binding of F} is lipid) [97], in yeast subunit 6, a 259-residue protein
point mutations [12,105- 112]. The mutational assembled and thus protected from degradation. altered [109]. The bound ATPase activity of F} is has been found to determine oligomycin sensitivity
events in genes for subunit a and b have not yet The situation is better defined in subunit e not inhibited by the mutant Fo, as one would of the ATP synthase complex [81] (Fig. 12). This
been identified. Several amino acid substitution in mutants. In two strains, the mutation interferes expect in analogy to the inhibition by DCCD- protein is homologous to subunit a of the E. eoli
the subunit e gene have been determined by amino with the integration of the polypeptide into the modified Fo [114]. This is partieularly puzzling in ATP synthase. Interestingly, the mutations in sub-
acid sequence analysis of the mutant protein or membrane. This is due to a substitution of Leu-31 the case of mutant strains DG 18/3 exhibiting an unit 6 are located close to the small conserved
nucleotide sequence analysis of the mutated gene by phenylanaline (une E 463) and Gly-23 by exchange of the DCCD-reactive Asp-61 by an region of the protein.
[107- 111]. Remarkably, most of the subunit e aspartic acid (une E429). [111]. The mutant sub- asparagine [109]. This minimal alteration leads to Four different amino acid exchanges leading to
mutations are dominant over the wild-type allele unit e containing Phe-31 is forced into the mem- the impaired binding of an F} whose ATPase oligomycin resistanee have been described for the
[106,113]. A few subunit emutations, and all brane, and a functional FoF1 originates, if the activity remains fully active. In contrast, if F1 is yeast and three for N. erassa (proteolipid) [97,116].
analysed mutations in genes for subunit a and b, mutant gene is present at high copy number. This rebound to wild-type Fo whose Asp-61 has been All amino acid exchanges reside in the C-terminal
are recessive [12]. The dominant character of many is not true for mutant subunit e containing Asp-23, modified by DCCD, the ATPase aetivity is in- hydrophobie stretch and are clustered around the
subunit e mutants is explained most probably by i.e., a highly polar carboxyl group [111]. hibited [114]. Thus, even subtle changes in 'func- invariant DCCD reactive glutamic (aspartic) re-
negative complementation [113]. Fo contains 6- 10 Of particular interest is the amino acid substitu- ti on al' residues lead to ehanges in the architecture sidue. It is a distinct possibility, that the amino
subunit e polypeptides which function in an inter- tion of Ala-21 by valine (DG 27/10) (Hoppe, J., of the whole complex. This structural or eonfor- acid residues altered in the resistant mutants are
dependent way. Thus, in a diploid situation a Schairer, H.U. and Von Meyenburg, K., unpub- ma tional effeets are further indicated by the ob- involved in the binding of oligomycin. Thus, these
mutated subunit e, which is still assembled into Fo, lished data). This mutant subunit e is integrated servation (Friedl, P., unpublished data) that sub- residues might be located at the lipid/protein in-
will inactivate most part of the available wild type into the membrane. The mutation is recessive, thus unit b is more easily extracted from Fo of mutant terphase. Some of them result in a hypersensitivity
subunit e. Accordingly, such dominant mutations the mutant subunit e is apparently not assembled strains DG 18/3 and DG 7/1 than from wild-type towards DCCD. Both facts suggest that the bind-
in subunit e do not interfere with the assembly into Fo (see above). In another mutant, the subunit Fo· ing sites for DCCD and oligomyein are overlap-
process, whereas the recessive mutations ap- e, exhibiting an exchange Pro-64 by leucine is ing.
parently are defective in assembly. Negative com- present in the membrane [2]. The mutant allele IVC-2. Inhibitor-resistant mutants
plementation should occur either not at all or to a shows a partial dominance over the wild-type al- Dicyclohexylcarbodiimide-resistan t m u tan ts V. Functional aspects
lesser extent with mutations in genes for subunits lele. This suggests that this mutation does allow have been isolated from E. eoli. Six mutant strains
a and b, which exist in Fo as single or dimeric the assembly into Fo [106,111]. have been described in detail [107,115]. Measure- VA. Euolutionaryaspeets
copies, respectively. Mutants DG 7/1 and DG 18/3 are both af- ment of the inhibition of ATPase aetivity as weil
In some of the compiled mutant strains, the fected in the invariant DCCD-reactive aspartic as ATP-dependent H+-translocation at various In recent years, numerous proteolipid subunits
affected subunit is not present in the membrane. residue 61 [107- 110]. These mutations are domi- eoncentrations of DCCD revealed two classes of from various organisms have been sequeneed, as
No direct information is available on the subunit a nant, and both mutant proteolipids are present in mutant strains [115]. Whereas the wild-type en- extensively reviewed elsewhere [6]. The proteins
mutant. This extremely hydrophobic protein could the membrane [113]. Thus, these alterations allow zyme is inhibited half-maximally at 3- 5 nmol contain 70- 82 residues and all may be purified by
not be analyzed by two-dimensional gel electro- an assembly of the mutant protein into Fo. DCCD per mg of membrane protein, the enzyme chloroform/ methanol extraction. They are ex-
phoresis, and antibodies are not yet available. The membranes of all Fo mutants compiled in from the class-one and class-two mutants are in- tremely hydrophobic, eontaining only 16- 25%
Effects in subunit b mutants are difficult to inter- Table IV are tight for protons with the exception hibited half-maximally only at 30 and 200 polar residues, whieh explains their solubility in
pret at the moment. The membrane of atp F of une F mutant DG 25/9. Thus, these mutations nmoljmg, respectively. DCCD at higher con- organic solvents.
mutant DG 25/9 contains subunit b, as de- interfere with the H + conduction catalysed by Fo. centration still binds specifically to subunit e as Long hydrophobie stretehes of about 25 amino
termined by immunological techniques (Friedl, P. , This applies for mutarits where the assembly of Fo revealed by sequenee analyses. For type one, Ile-28 acid residues are found both in the N-terminal as
unpublished data). This mutant has a leaky pheno- is defective. The phenotype of these point mutants was substituted by valine. In type two mutant weil as in the C-terminal part of the sequence. This
type, since ATP-dependent protontranslocation is corresponds to that of deletion mutants, whieh protein, a threonine was found at position 28. is reminiscent of the most thoroughly investigated
partially retained [105]. A similar phenotype has have been discussed in detail above (Table III). Most likely, Ile-28 affected in all resistant mutants membrane pro tein, the baeteriorhodopsin from
been described recently for another atp F mutant But it applies also to those mutants where the is part ofthe DCCD-binding site. Enzymic activi- Halobaeterium halobium, where stretehes of 25 hy-
[113]. In strain une F 469 subunit b was not mutated subunit apparently is assembled into Fo. ties, sueh as ATPase activity and ATP-dependent drophobic amino acids are found to traverse the
detected when whole membranes were analysed by Of particular interest is the question of whether H + translocation, of these mutants are comparable lipid bilayer most probably in an a-helieal confor-
two-dimensional gel electrophoresis [12]. Since the binding of the F}-ATPase is influenced by ~o .the wild-type protein. These results provide mation (Fig. 11).
subunit b is extremely prone to proteolytic de- mutations in Fo subunits. The binding affinity of mdlrect evidence that the protein folds baek A comparison of the sequences of the homolo-
gradation, as described above, it is uncertain F} has not yet been quantitatively analysed. This bringing Ile-28 and Asp-61 into proximity. ' gous proteolipid subunits revealed numerous con-
whether the mutation prevents integration into the may explain discrepancies reported for the rebind- The analysis of oligomycin-resistant mutant served amino acid residues. These residues might
membrane or whether a defective assembly results ing of F1 to Fo from certain mutants by different from S aeeharomyees eereuisiae and N. erassa be indispensible for function or the maintainance
in a proteolytic degradation. Tentatively, it may be groups [102,106]. Despite these uncertainties, the revealed that two proteins may be involved in the of a certain structure of the protein. In the middle
concluded that the latter strain is assembly-defec- most surprising result is that in mutant strains inhibitor binding. Besides the subunit e (proteo- of the N-terminal hydrophobic stretch four glycine
16 17
TABLE V teolipid subunit (subunit c). For the proteolipids But it could not be excluded that the increased
Iso VALUES FOR SOME DCCD ANALOGUES extracted with butanol from lettuce chloroplasts or conductivity at higher pH results from an in-
R-N C=N-R', R=cyc\ohexyl. Iso values for the ana- yeast mitochondria, a proton permeability of re- creased proton permeability of a single channel.
logues were obtained using incubation for 16 at O°C at various constituted lipid-proteolipid bilayers has been In the reported experiments, the pH induced
inhibitor concentrations by measuring either ATPase activity or demonstrated [129-132]. conductance transition was only found in the pres-
A TP-dependent H + translocation. Recently, single-channel conductance has been ence of cholesterol and sufficiently high proteo-
W 1',)
measured after reconstitution of the proteolipid lipid concentrations. The interaction between pro- W
0:
u.. " .. '" I 1 1 111 1 I 1
Compound R Iso at R' 11: III! ! III! 11
from yeast into planar lipid bilayers [133]. Thus, in teolipid subunits therefore may not be sufficiently 1
(nmoljmg l
membrane contrast to earlier studies, proton conductivities strong to stabilize proton-conducting proteolipid
protein) could be measured at a high-time resolution as a oligomers in a biological membrane. I
0"
teinases, and thus located at the cytoplasmic J., Brunner, J. and Jorgensen, B.B. (1983), unpub- remains uneertain whether the observed [125 I]TID
surface [49]. Taken that seven transmembrane seg- lished data). labelling pattern represents the same modification ,flflfUif
ments exist, this would indicate that the C-termi- Labelling with the freely mobile carbene-gener- of each of the subunits or the sum of two different
nal end would be located on the periplasmic side. e
ating probe 25 I]TID started very closely to the labeIling patterns. Several of the residues not at-
The proteolytical breakdown products of subunit a N-terminus at Leu-3 and ceased at Trp-26. With a taeked by [125 1]TID have hydrogen-bonding capae-
are difficult to analyze, even with membranes from nitrene-generating probe fixed to the polar ity (Asn-2, Thr-6, Gln-10, Lys-23, Tyr-24). These ~ 1''> LIPID PHASE
an ATP synthase overproducing strain, due to the headgroups of a phospholipid (' shallow probe'. residues might be involved in contacts with other E ! I
presence of many additional proteins. But at raised Fig. 14), residues Asn-2 as weil as Cys-21 and subunits. But it could weIl be that a microenviron- G I~QP A DA
.. ,l N E
concentrations of proteinases the amount intact of Trp-26 were modified. Thus, the entire N-terminus ment created by these side-ehains does not allow
DCCO R UNCB=~~l
subunit a in the membrane decreases, indicating up to Trp-26 is embedded in the membrane and the aecumulation of [ 125 I]TID at these positions
that other parts of the polypeptide chain are acces- the boundaries are defined 'by the residues affected and thus prevents an efficient modification.
sible to proteinases. It is conceivable that polar by the 'slrallow probe'. Most likely, the N-terminal The large polar domain of subunit b is clearly Fig, 15. Prediction of secondary structures and membrane-per-
segment domains of subunits exposed at the cyto- segment traverses the whole phospholipid bilayer exposed at the cytoplasmic side of the membrane, meating segments in Fo subunit e of E. eoli. a-helical segments
(~) and ß-turns (T) were consistently ca1culated applying four
plasmic surface of the membrane are involved in in an a-helical conformation [134,135]. But the since it can be eompletely degraded with pro-
different prediction methods [77-80], The free-energy gains
the binding of F1 as was observed in mutant other possibility, that the chain folds back and teinases [49]. The two molecules of subunit b in Fa during a transition from a random coil in water to an a-helix in
strains containing only this Fa subunit. that Asn-2 and Trp-26 are located at the same can be efficiently cross-linked (see Fig. 9). They the membran es were ca1culated for all amino acid sequence
boundary, is not excluded by the available experi- exist, therefore, as a dimer. Most likely, the con- positions using the parameters given by Von Heijne [134].
VIB. Subunit b mental results [136]. At positions 27 and 28, two taet is formed by the large polar domain. This
proline residues are located which will not fit into large polar domain binds F1 as demonstrated in
The polarity profile of the sequence of 151 an a-helix, and, consequently, their peptide bonds mutant membrane where the other Fa subunits a
residues of subunit b is striking in that about 30 can genera te a rather large hydrophilic surface and c are deleted [102]. This is not the only contact philic segment, is found to be conserved in the
hydrophobic amino acids are clustered at the N- area. It is thus reasonable that the polypeptide side between Fa and F1 , since - as already men- homologous subunits from other bacteria, as weil
terminus (Fig. 13), whereas the rest of the poly- chain leaves the membrane at this point. tioned - subunit a also is involved in the binding as from mitoehondria and chloroplasts. This clus-
peptide chain is very polar similar to a water-solu- Surprisingly, most of the N-terminal residues of F1 [102]. In cross-linking experiment, dimers of tering of hydrophobic and hydrophilic residues
ble protein. This immediately suggests that the were accessible to the small diffusables probe subunit b with F1 subunits a and ß are observed. A immediately suggested that the pro tein might
N-terminus of subunit b is integrated in the mem- [
125
1]TID. This demonstrates that this segment is close interaetion of the F1 subunits a and ß with traverse the membrane twiee in a hairpin-like
brane. In fact, all applied hydrophobic photoreac- not buried in a core of Fa but rather is located at subunit b is also indicated by the observation that structure. Indeed, labelling experiments with
125
tive probes reacted exclusively with this hydro- the periphery. As two subunits b exist in one Fa, it in eertain mutant strains subunit b is protected [ 1]TID indicate both hydrophobie segments
phobic segment (Refs. 71 and 72, see also Hoppe, against proteolytic degradation in vivo by the F 1- being loeated in the lipid bilayer. Especially intri-
subunits a andjor ß, even in the absence of the guing is the location of the acidic residue Asp-61
subunits y, 0, and ( [49]. Interestingly enough, the in the membrane, as demonstrated by its reactivity
polar domain of subunit b contains internal re- towards only hydrophobie carbodiimides. Re-
peats [29]. Residues 53-82 are clearly homologous markably, an acidic group is not tolerated in the
with residues 85-105. Furthermore, a shorter re- N-terminal segment at position 23 of the E. eoli
peat is found, residues 84-98 are homologous to protein, as seen with subunit c from a mutant une
6 residues 101-113 [29]. Thus it can be visualized E 429, (see Table IV) which is not integrated into
'"zw
w that the subunit b dimer contains four roughly the membrane. Only distinet residues of the sub-
w
w
'"
IL
equivalent domains which might interact with the unit e are aecessible to the label [125 I]TID in
multiple copies of subunits a and ß present in F1 . functional Fa. In certain special cases, the nonla-
Point mutations in subunit b (see Table III) belling by TID might be explained by a low reac-
[105,112] result in a weakened binding of F1 • Un- tivity of the side chain (e.g., alanine; see Hoppe, J.,
fortunately, the mutated amino acid residue has Brunner, J. and Jsbrgensen, B.B. (1983), unpub-
Fig. 13. Prediction of membrane-permeating segment and sec- not yet been identified. lished data) or by the instability of the re action
ondary structures in the 1'0 subunit b from E. co/i. a-helical product (e.g., aspartyl ester). But eertainly, in most
regions (l1e) and ß-turns (T) were consistently predicted by four VIC. Subunit e eases residues are not labelIed, since they are buried
different prediction methods [77-80]. The free-energy gains for in the interior of the quarternary structure of Fa. It
a transition from a random coil in water to an a-helix in the Fig. 14. A possible arrangement of the N-terminus from sub-
The polarity profile of the amino acid sequence may be relevant in this conneetion that a mutant
membrane was calculated for all amino acid sequence positions unit b in the lipid bilayer. Shaded amino acid symbols indicate
using the parameters given by Von Heijne [134]. The location labelling with TID. Hatched areas indicate labelling with the of the E. eoli subunit e (see Fig. 15), i.e., two proteolipid from E. eoli, whieh is unable to assem-
of acidic (.j,) and basic ( t ) residues is indicated by arrows. 'shallow probe'. hydrophobie segments interrupted by a hydro- ble (see Table IV), has an amino acid substitution
22 23
of Ala-21 by valine in a segment not accessible to have not been performed with the mitochondrial tion of this residue (Asp ~ Asn) leads to impaired [137]. An alternative would be that the oligomer of
TID. proteolipids. Thus, it is uncertain whether corre- binding of F1 (see chapter IV C-l) and the Fo subunit c binds water either by the amino acid
Possibly, the small side-chain of the alanine at sponding positions in the bacterial or mitochondri- subunit b. Apparently, large conformational side-chains or by the polar groups of peptide
position 21 is part of a contact site in the tertiary al proteolipids are equallly accessible from the changes are induced in the whole Fo by this muta- bonds. In many instances, buried water clusters
structure of the proteolipid or the quarterly struc- lipid phase. It has to be stated, however, that in an tion. were found in the interior of globular proteins. For
ture of Fo, and, therefore, no larger side-chain is a-helical confirmation, so me mutated residues example, in studies with chymotrypsin, it was dis-
tolerated at this position. The pattern of [125 I]TID- would point in opposite directions. VID. Models JOl' proton eonductanee cussed that each region associated with buried
labelIed residues cannot be unambiguously inter- The various positions determining the oligomy- charged groups contained an extensive hydrogen-
preted at the moment, since the labelling of in- cin resistance all are located in the vicinity of the All available evidence points to the proteolipid bonded network including several buried water
dividual subunit c molecules in the Fo might be DCCD-reactive acidic residue in the C-terminal subunit as the protonophoric entity of the Fa. molecules [138]. Recent X-ray studies on
different. It is conspicuous, however, that at the segment [97]. In contrast, only one position de- Several lines of evidence argue against a direct alamethicin, which forms an oligomeric voltage-
N-terminal segment the labelIed residues would termining DCCD resistance in the E. coli proteo- participation of subunits a and b in transmem- ga ted ion channel, shed light on the possible in-
mark one side of a continuous a-helix. Also in the lipid, i.e., Ile-28, was found in the N-terminal brane protontranslocation. The large hydrophilie volvement of bound water moleeules in cation
C-terminal part the labelIed residues could be segment [115]. In a hairpin-like structure of the part of subunit b might be involved in the coupling transport [139]. The alamethicin monomer forms
located on a-helical segments. proteolipid, this residue would be located in between the proton translocation across the mem- an helix which has abend in the middle due to the
It has been discussed previously that certain proximity to the carbodiimide reactive aspartic brane and ATP synthesis/hydrolysis on the F1 , occurrence of a proline residue. This proline-in-
residues of the proteolipid altered in oligomycin- residue. It is likely that Ile-28 is involved in nonco- but it is unlikely, that the short uncharged NH 2 - duced bend results in two free carbonyls and one
resistant mutants from yeast and Neurospora are valent binding of e.g., the cyclohexyl moiety of terminal segment, which has only loose contact free amide group of the peptide backbone, thus
accessible from the lipid phase. It was therefore DCCD. Surprisingly enough, Ile-28 is not accessi- with the other subunits, provides residues par- providing a hydrophilic surface (Fig. 17). It was
interesting to examine whether these residues cor- ble to TID in the functional Fo. Differences in ticipating in the mechanism of H+ conduction. If visualized that this area, together with a buried
respond to the TID accessible residues in the E. sterical factors between TID and DCCD are dif- subunit a contains the proton pathway, then only glutamine residue, is able to bind a large number
eoli proteolipid. Three amino acids altered in the ficult to evaluate, but are not a likely explanation the few conserved residues may be considered of water molecules. A corresponding water-bind-
proteolipid from inhibitor-resistant mutants occur for the different accessibility of Ile-28 for both which are clustered in a short segment. For sub- ing structural element might be present in subunit
at positIOns coinciding or juxtaposed to compounds. It is a distinct possibility that either unit e Schindler and Nelson [133] provided con- e. In the middle of the C-terminal hydrophobie
TID-labelled residues from the E. coli proteolipid DCCD is bound via an 'induced fi t' or that DCCD vincing evidence that the isolated protein func- segment a proline residue is found in all bacterial
(Fig. 16). In two instances, however, the mutated binds only to a certain conformation of the Fo tions as a protonophore, when reconstituted in proteins. In the mitochondrial and chloroplast
residue is located two positions apart. occurring with a low probability. black lipid membranes. These in vitro experiments proteolipids generally a helix-breaking residue
Unfortunately, [125 I]TID labelling experiments The experimental data discussed above are rele- seem to contradict results obtained with mutant (threonine, glycine) exists at the corresponding
vant with respect to the structural properties of the membranes from E. eoli, which contain only the position. This bend is located four residues apart
proteolipid. In order to elucidate the mechanism Fo-subunit e and which are unable to conduct from the membrane buried acidic residue Asp-61.
of the proton conduction, it is also important to protons [102]. This discrepancy might be resolved
~
identify functional residues - i.e., residues directly by the observation of Schindler and Nelson that
involved in proton conductance. It has been sug- the active proton channel formed in black lipid
~,
gested that Asp-61 in the E. coli proteolipid is such membranes is at least a dimer and probably a
. v.•·.
L.:.'·M a functional residue. (All structural evidence indi- high er oligomer of the subunit c. In vivo this
YA~
....•Y
~
cates this residue being located in the interior of functional oligomer might be stabilized by the Fa
the membrane.) Its selective modification with subunit a and/or b as originally proposed by
Y.L/
J>;
M 0 ··.·N
. DCCD abolishes proton conductance. The impor-
tance of this residue was further indicated by the
Nelson and Schatz [92].
Therefore, at present any discussion on the
~
N L
0/
E
observation that this acidic residue is strictly con-
served in the proteolipids from mitochondria,
bacterial and chloroplasts. Futhermore, proton
mechanism of proton conductance will con-
centrate on a subunit c oligomer. However, based
on the presently available structural data, the
Fig. 16. Areas of subunit e from the E. eoli ATP synthase which conduction is lost if this residue is genetically mechanism of proton conductance is completely
are exposed at the lipid phase and positions of amino acid altered. obscure. The membrane spanning segments of
exchanges in oligomycin-resistant mutants from yeast and N. Some observations, however, are difficult to subunit c contain only a few polar residues and
e/'a5'sa. A planar representation of the two putative a-helices at only one charged residue - the invariant DCCD-
reconcile with a purely functional role of this
the N-terminus and the C-terminus is shown. Shaded areas
indicate residues possibly in contact with lipids as revealed by acidic residue. This residue is accessible from the reactive aspartic acid. Therefore, it appears dif-
Fig. 17. Backbone hydrogen bonding in alamethicin around
labelling with [ 125 I]TID. Arrows indicate positions where amino lipid phase and is therefore most likely not located ficult to construct a network of hydrogen bonds proline-14 [139]. The arrows indicate free carbonyl or amide
acid substitution leading to oligomycin resistance was found. in the interior of apore. A minimal genetic altera- across the membrane from amino acid side-chains groups of peptide bonds.
24 25
However, the participation of this aCldlC resldue 111 The topology of all three Fo-subunits might be 19 Mabuehi, K., Kanazawa, H., Kayano, T and Futai, M. 47 Engelman, D.M. and Zaccai, G. (1980) Proe. Nat!. Acad.
the formation of a water cluster inside a pore is (1981) Biochem. Biophys. Res. Commun. 102, 179 Sei. U.S.A. 77, 5894-5898
elucidated in more detail by means of antibodies, 48 Sone, N., Yoshida, M., Hirata, H. and Kagawa, Y. (1978)
20 Kanazawa, H., Kayano, T., Mabuehi, K. and Futai, M.
difficult to reconcile with its reactivity to DCCD. directed against defined polar segments of the Proc. Nat!. Acad. Sei. U.S.A. 75, 4219-4223
(1981) Bioehem. Biophys. Res. Commun. 102, 604-612
The central question concerning all studies di- polypeptide chains. The amino acid sequences will 21 Kanazawa, H., Mabuchi, K., Kayano, F., Noumi, T, 49 Hoppe, J., Friedl, P., Sehairer, H.U., Sebald, W., Von
rected to structure and function is whether Fa can furthermore give hint for possible chemical mod- Sekiya, T. and Futai, M. (1981) Bioehem. Biophys. Res. Meyenburg, K. and Jorgensen, B.B. (1983) EMBO J. 2,
exist in different conformations. In the F1-ATPase, ification studies directed towards identification of Commun. 103, 613-620 105-110
extensive conformational changes occur upon en- 22 Kanazawa, H .. Mabuehi, K. and Futai, M. (1982) Bio- 50 Pedersen, P.O., Hullihen, J. and Wehrle, J.P. (1981) l Bio!.
functional residues or towards the accessibility of Chem. 256, 1362-1369
ehem. Biophys. Res. Commun. 107, 568-575
ergization of the membrane and during ATP hy- certain segments. 51 Loo, TW. and Bragg, p.D. (1982) Bioehem. Biophys. Res.
23 Kanazawa, H., Kayano, T, Kiyasu, T and Futai, M.
drolysis or synthesis [4]. It could weil be that Conformational changes might be defined by (1982) Biochem. Biophys. Res. Commun. 105, 1257-1264 Commun. 106, 400-406
conformational changes in F1 are accompanied or membrane permeating photoreactive probes and 24 Foster, D.L. and Fillingame, R.H. (1982) J. Bio!. Chem. 52 Hopp, TP. and Woods, K.R. (1981) Proe. Nat!. Acad. Sei.
even coupled to conformational changes in Fo. by the covalent attachment of reporter groups, i.e., 257, 2009-2015 U.S.A 78, 3824-3828
Papa and co-workers [140] provided experimental 25 Von Meyenburg, K., Jj6rgensen, B.B., Nielsen, J., Hansen, 53 Bayley, H. and Knowles, J.R. (1977) Methods Enzymo!.
spin labels or fluorescence label. Finally, it might 46,69-114
F. and Michelsen, O. (1982) Tokai J. Exp. Clin. Med., in
evidence for two different conformation states of be possible to obtain Fo-crystals sufficient for high 54 Chowdry, V. and Westheimer, F.H. (1979) Annu. Rev.
the press
the proton conductor in dependency of the applied resolution X-ray analysis. Hopefully, the results 26 Sebald, W., Graf, T and Lukins, H.B. (1979) Eur. J. Bioehem. 48, 293-325
ßf-tH+ in beef-heart mitochondria. Also, the inhibi- obtained by proteinchemical, physiochemical and Bioehem. 93, 587-599 55 Staros, J.V. (1980) Trends Biochem. Sei. 5, 320-322
tory action of oligomycin can be readily explained genetic methods then can be integrated into a 27 Sigrist-Nelsen, K., Sigrist, H. and Azzi, A. (1978) Eur. J. 56 Brunner, J. (1981) Trends Bioehem. Sei. 6, 44-46
if this macrocyclic compound induces or stabilizes Biochem. 92, 9-14 57 Bercovici, T and Gitler, C. (1978) Bioehemistry 17,
consistent picture.
28 Hoppe, J., Brunner, J., Friedl, P., Lincoln, D., Von Meyen- 1484-1489
one structural state. The same inhibitory mecha- burg, K., Michelsen, O. and Sebald, W. (1982) 2nd 58 Wells, E. and Findlay, J.B. (1979) Biochem. l 179,265-272
nism may apply for DCCD. Pleiotropic effects of References
European Bioenergetic Conferenee, Short report, pp. 85-86 59 Bayley, H. and Knowles, J.R. (1978) Biochemistry 17,
amino acid substitutions in subunit e (see subsec- 1 Downie, J.A, Gibson, F. 3nd Cox, G.B. (1979) Annu. 29 Walker, .J.E., Saraste, M. and Gay, N.J. (1982) Nature 298, 2420-2423
tion IVC) might be caused by allosteric interac- Rev. Bioehem. 48, 103-131 867-869 60 Brunner, J. and Semenza, G. (1981) Bioehcmistry 20,
2 Gibson, F. (1982) Proe. R. Soe. Lond. B 215, 1-18 30 Unwin, P.N.T. and Henderson, R. (1975) J. Mo!. Bio!. 94, 7174-7182
tion. A mechanism of proton translocation by a 3 Referenee deleted 425-440 61 Cerletti, N. and Schatz, G. (1979) J. Bio!. Chem. 254,
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