Role of the N- and C-terminal regions of the PufX protein in the structural organization of the photosynthetic core complex of Rhodobacter sphaeroides

Role of the N- and C-terminal regions of the PufX proteinin the structural organization of the photosynthetic corecomplex of Rhodobacter sphaeroides

Francesco Francia1,2, Jun Wang1,*, Hans Zischka1,†, Giovanni Venturoli2 and Dieter Oesterhelt1

1Department of Membrane Biochemistry Max-Planck-Institute for Biochemistry, Martinsried, Germany; 2Department of Biology,

Laboratory of Biochemistry and Biophysics, University of Bologna, Italy

The core complex of Rhodobacter sphaeroides is formed bythe association of the light-harvesting antenna 1 (LH1) andthe reaction center (RC). The PufX protein is essential forphotosynthetic growth; it is located within the core in a 1 : 1stoichiometry with the RC. PufX is required for a fastubiquinol exchange between the QB site of the RC and theQo site of the cytochrome bc1 complex. In vivo the LH1–PufX–RC complex is assembled in a dimeric form, wherePufX is involved as a structural organizer.Wehavemodifiedthe PufX protein at the N and the C-terminus with pro-gressive deletions. The nine mutants obtained have beencharacterized for their ability for photosynthetic growth, theinsertion of PufX in the core LH1–RC complex, the stabilityof the dimers and the kinetics of flash-induced reduction ofcytochrome b561 of the cytochrome bc1 complex. Deletionof 18 residues at theN-terminusdestabilizes thedimer in vitro

without preventing photosynthetic growth. The dimer (or astable dimer) does not seem to be a necessary requisite forthe photosynthetic phenotype. Partial C-terminal deletionsimpede the insertion of PufX, while the complete absence ofthe C-terminus leads to the insertion of a PufX proteincomposed of only its first 53 residues and does not affect thephotosynthetic growth of the bacterium. Overall, the resultspoint to a complex role of the N and C domains in thestructural organization of the core complex; the N-terminusis suggested to be responsible mainly for dimerization, whilethe C-terminus is thought to be involved mainly in PufXassembly.

Keywords: LH1-RC; photosynthesis; PufX; Rhodobactersphaeroides.

The purple bacterium Rhodobacter (Rb.) sphaeroides cangrow photosynthetically or heterotrophically via aerobic oranaerobic respiration. When growing photosynthetically, ituses light energy as a driving force to form ATP via a cyclicelectron transfer. Photons are captured from the light-harvesting (LH) complex(es) and the excitation energyfunnelled towards a bacteriochlorophyll (BChl) special pair(P), located in the reaction centre (RC). The excited Pdelivers an electron via an accessory BChl and a bacterio-pheophytin molecule to a primary ubiquinone acceptor(QA). In amuch slower reaction the electron is transferred toa second ubiquinone acceptor (QB). The full reduction of

the quinone molecule at QB to quinol requires a secondphotoexcitation of the RC and is coupled to the uptake oftwo protons from the cytoplasmic space. The formedubiquinol dissociates from the RC and is released into themembrane lipid phase [1]. Ubiquinol molecules are oxidizedat the Qo site of the cytochrome bc1 complex (cyt bc1). Herethe electron pathway branches into a high and a lowpotential chain. The first electron reduces in series an ironcluster centre and a cytochrome c1 in the high potentialchain, while the second electron reduces the low potentialchain, composed of cytochrome b566, cytochrome b561 anda ubiquinone molecule located at the Qi site. A secondubiquinol oxidized at the Qo site brings the electron to fullyreduce the ubisemiquinone to ubiquinol on Qi. From thecytochrome c1, the electron is transferred to a solublecytochrome c2 that is the physiological electron donor to theoxidized P. From the Qo site, protons are released into theperiplasmic space of the cell. This cyclic mechanism of redoxreactions acts as a proton pumping system, moving protonsfrom the cytoplasmic to the periplasmic space. The formedH+ gradient is the driving force for synthesis of ATP that isused to power the metabolic reactions in the cell [2].

In Rb. sphaeroides the ability of the RC to capture lightenergy is largely increased by the presence of two LHcomplexes: LH1 and LH2. The LH1 complex is intimatelyassociated with the RC in a fixed stoichiometry to form thecore complexes (LH1–RC), while the LH2 is arrangedperipherally with respect to the core. Both LHs areorganized in circular supramolecular complexes, resultingfrom the repetition of a minimal building block commonly

Correspondence to F. Francia, Department of Biology, Laboratory of

Biochemistry and Biophysics, University of Bologna, Via Irnerio n.42,

40126 Bologna, Italy. Fax: + 39 051 242576,

Tel.: + 39 051 2091300, E-mail: [email protected]

Abbreviations: BChl, bacteriochlorophyll; cyt bc1, cytochrome bc1complex; ICM, intracytoplasmic membranes; LH, light-harvesting

complex; PMC, photosynthetic membrane complex; QA,QB, primary/

secondary electron acceptor; Qi, quinone reductase site of the cyt bc1complex; Qo, quinol oxidase site of the cyt bc1 complex; RC, reaction

center.

*Present address: Department of Plant Biology, The Ohio State Uni-

versity, Columbus, OH, USA.

Present address: GSF Forschungszentrum, Institut fur Humangene-

tik, Oberschleißheim, Germany.

(Received 6 November 2002, revised 12 February 2002, accepted 13

February 2002)

Eur. J. Biochem. 269, 1877–1885 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02834.x

referred to as the a,b heterodimer. The a and b polypeptidesspan the membrane with a single hydrophobic a helix. Thiscircular protein scaffold binds the pigments that aremaintained in a spatial orientation that maximizes theefficiency of the energy transfer reactions.

Structures of the LH2 [3,4] and RC [5], as well asof cyt bc1 [6] are known at atomic resolution but camefrom different organisms; on the contrary, high resolu-tion structural data of the core complex are not yetavailable.

In Rb. sphaeroides and Rb. capsulatus photosyntheticgrowth requires the presence of the PufX protein [7,8].When an intact LH1–RC core complex is present, PufX isessential to promote an efficient ubiquinone/ubiquinolexchange between the RC and cyt bc1 [9], but is notnecessary when the LH1 system is absent or reduced in size[10–12]. This evidence points to a complex structuralrelationship between the components of the photosyntheticsystem, in which PufX plays a central role [13]. Recently,several works have indicated that PufX is involved directlyin the supramolecular organization of the photosystem: (a)the core complexes of Rb. sphaeroides are organized in adimeric form [14], in which the presence of PufX induces aspecific orientation of the RC inside the LH1 complex aswell as the formation of a long range regular array of LH1–RC in the photosynthetic membrane [15]; (b) biochemicalstudies have shown that PufX is present in the LH1–RCcomplex in a 1 : 1 stoichiometry with the RC, and that thedimeric form of the core complex could only be isolated inthe presence of PufX [16]; (c) the PufX protein has a strongtendency to interact with the LH1 a polypeptide, while nointeraction was detected with the LH1 b polypeptide [17];(d) the deletion of PufX increases the number of LH1-associated Bchls per RC, suggesting an increased number ofa,b heterodimers in the LH1 [18]. Moreover in the presenceof PufX, electron density maps of the dimeric LH1–RCshow unequivocally interruptions in the LH1 ring encirclingthe RC. The top view of the LH1–RC core complexpresents two rings of LH1 in close contact forming a patternwhich resembles the shape of the letter S; each interruptedring contains an electron dense nucleus attributed to the RC[14].

All of these experimental results are consistent with theidea that PufX is responsible for these interruptions,allowing a faster lateral diffusion of ubiquinone/ubiquinolmolecules toward/from the RC QB site.

A previous work on Rb. sphaeroides demonstrated therole of the C-terminal amino-acid residues of the LH1 apolypeptide in the organization of the LH1–RC complex[12]. In the present work, we have investigated thepossible involvement of the N-terminus and of theC-terminus of PufX in protein–protein interactions stabil-izing the LH1–RC complex. To this aim two sets ofmutant strains have been constructed. The N-terminaldomain has been progressively shortened by deletionsextending from the second residue of the primarysequence, while the C-terminal portion has been progres-sively shortened by introducing stop codons by site-directed mutagenesis.

We have obtained information on the involvement of theN- and C-terminal portions of PufX in its insertion in themembrane and dimerization of the core complexes.

E X P E R I M E N T A L P R O C E D U R E S

Bacterial strains, plasmid, gene transfer, growthconditions, membrane preparations

Bacterial strains and the plasmid used in this work were asdescribed previously [16]. Growth conditions forEscherichiacoli andRb. sphaeroides have also been described [16]. All ofthe Rb. sphaeroides strains were grown semi-aerobically.Photoheterotrophic growth tests in liquid culture weremonitored with a Klett–Summerson colorimeter asdescribed by Farchaus et al. [7]; kanamycin and tetracyclinewere added at 25 lgÆmL)1 and 2 lgÆmL)1, respectively.Cultures were illuminated by two 120 W incandescent lightbulbs; excessive warming was prevented by placing a 40-cmwater bath between the lamps and the cultures. Intra-cytoplasmic membranes (ICM) were prepared as describedpreviously [19].

PufX mutagenesis

The PufX N-terminal deletion and C-terminal stop codonseries were constructed using the pRKX plasmid [16] asDNA template and introducing the desired mutation/deletion with the method given by Ausbel et al. [20]. Theexternal primers used for the cited sequential PCR muta-genesis anneal, respectively, 330 bp upstream and 441 bpdownstream the pufX gene on the plasmid. This fragmentcontains the HindIII and ClaI (8 bp upstream and 171 bpdownstream the pufX gene, respectively) unique sites andallows, after digestion withHindIII andClaI, the ligation ofthe final PCR product containing the mutated pufX genewith the pRKX vector digested with the same restrictionenzymes. After transformations of E. coli S17-1 cells, singlecolonies of the putative transformants were grown over-night in 5 mL Luria–Bertani media with 10 lM tetracycline.The pRKX derived harbouring mutated pufX gene con-structs from the E. coli S17-1 cells were introduced intoRb. sphaeroides DQ x/g cells by conjugation [16]. Muta-tions were confirmed by sequencing the plasmids isolatedfrom transformed Rb. sphaeroides cells with the QiagenMinikit.

Isolation of core complexes and SDS/PAGE

Core complexes were extracted from (ICM) according tothe method described previously [16,21] except that theNaBr washing step was performed at 0.6 mgÆmL)1 totalprotein.

The concentration of LH1–RC complex in the isolatedbands was estimated on the basis of the total photooxid-izable RC measured by flash kinetic spectrophotometry asdescribed before [16]. Aliquots containing the same numberof LH1–RC moles were treated with 10 vol. cold acetone/methanol (7 : 2, v/v), vortexed for 2 min and centrifuged.The organic phase was discarded, and the protein pellet wasdried at 40 °C for 30 min. The pellets were redissolved inSDS/PAGE loading buffer to final concentration of 2 lMLH1–RC in all of the samples.

SDS/PAGE was carried out accordingly to Schagger &Von Jagow [22], with a separating gel of 19.5% (w/v)acrylamide, 0.5% (w/v) bis-acrylamide.

1878 F. Francia et al. (Eur. J. Biochem. 269) Ó FEBS 2002

MS and sequencing of PufX54* protein

Proteins were separated by SDS/PAGE as described above,and stained with Coomassie G250.

Gels were washed extensively with H2O to removeresidual acid from the destaining process. The band ofinterest was cut out with a razor blade and transferred toreaction tubes. Proteins were then subjected to a limitedprotease treatment overnight (0.5–1 lgÆband)1 endopro-teinase LysC, Roche Molecular Biochemicals) [23].

Peptides were extracted from gel slices by alteredincubation with 10% formic acid and acetonitrile. Pooledfractions were dried in a speed vac concentrator. Driedpeptides were re-dissolved in 10 lL 10% acetonitrile/0.1%trifluoroacetic acid. Between 0.5 and 1 lL were used forMALDI-TOF analysis (adapted from [24]).

The residual sample was applied to a reversed-phaseHPLC system to separate peptides. Purified peptides weresubjected to automated Edman degradation (with kindsupport of J. Kellermann, Max Planck Institute forBiochemistry, Martinsried).

Time resolved spectroscopy on ICM

The kinetics of cytochrome b561 reduction induced by asingle actinic flash were measured under the followingconditions: ICM were resuspended in a buffer composed of50 mM Mops, 100 mM KCl, pH 7.0; valinomycin andnigericin were added at 10 lM to collapse the transmem-brane proton gradient and to avoid spectral interference dueto BChl and carotenoid electrochromic effects; 5 lMantimycin A was used to inhibit the Qi site of the cyt bc1.Measurements were performed in a nitrogen atmosphere

under controlled redox conditions as described by Venturoliet al. [25]. One micromolar each of phenazine methosulfateand phenazine ethosulfate; 2 lM of 2,3,5,6-tetra-methyl-p-phenylenediamine; 10 lM each of p-benzoquinone, duro-quinone, 1,2-naphthoquinone, 1,4-naphthoquinone wereused as redox mediators. The experimental apparatus is asdescribed in Francia et al. [16].

Traces of cytochrome b561 reduction (Fig. 3) were ana-lysed numerically in terms of pseudo first-order kineticsfollowing an initial lag period, as described by Barz et al. [9].In order to determine the best fitting parameters, the lagperiod following the time of the flash was varied stepwise:for each lag period the amplitude and rate constant of theexponential function were optimized using a nonlinear v2

minimization routine [26] and a plot of the minimized v2 vs.the lag period was constructed for each kinetic trace. For alltraces this procedure yielded a minimum reduced v2 (v2

min)between 0.8 and 1.2. The confidence interval in thedetermined value of the lag was obtained by using anF-statistic to determine the probability p of a particularfractional increase in v2 according to:

v2=v2min ¼ 1 þ ½m=ðn ÿ mÞ�Fðm; n ÿ m; 1 ÿ pÞ

where m is the number of parameters, n is the number ofdata points, and F is the upper (1–p) quantile for Fisher’sF distributions with m and (n–m) degrees of freedom [27].Confidence intervals within 1 SD (P ¼ 0.68) calculated bythis procedure are given in Table 2. These intervals are

generally asymmetrical, due to the nonlinear nature ofdeconvolution.

R E S U L T S

Construction of the PufX N-terminally deleted seriesand C-terminally truncated series

To obtain the strains with the mutated PufX proteinreported in Table 1, two sets of plasmids were constructed.The first series consists of a progressive deletion at theN-terminus, extending from the second residue of theprimary sequence; the second series consists of a progressivetruncation of the C-terminal domain of PufX (Fig. 1)obtained by the introduction of stop codons in the genesequence of pufX. In all the cases, the Rb. sphaeroides hoststrain was DQ x/g [10].

The pseudo wild-type strain used in this work wasobtained reintroducing the complete puf operon via theplasmid pRKX (in trans) into the host Rb. sphaeroides DQx/g, deprived of the chromosomal copy of the puf operon.

Table 1. Bacterial strains and plasmids. The plasmid host strain in all

the cases was Rb. sphaeroides DQ x/g.

Strain Plasmid name

Wild-type pRKX

N-Terminus series

PufXD2–4 pRKXD2–4PufXD2–7 pRKXD2–7PufXD2–19 pRKXD2–19PufXD2–26 pRKXD2–26C-Terminus series

PufX54* pRKX54*

PufX68* pRKX68*

PufX72* pRKX72*

PufX76* pRKX76*

PufX81* pRKX81*

PufDX pRKDX

Fig. 1. Nature of the deletions and truncations on PufX. The helix

transmembrane region of the PufX protein, predicted with the program

PHDHTM [32], is indicated at the top of the figure and represented as an

empty rectangle in the primary sequences of PufX showed below. The

related Rb. sphaeroides strains are given on the left.

Ó FEBS 2002 Core complex organization in Rb. sphaeroides (Eur. J. Biochem. 269) 1879

Photosynthetic growth curves

Aliquots of bacteria corresponding to 1 absorbance unit at700 nm from precultures grown semi-aerobically in dark-ness were transferred to 13 mL final volume of fresh mediain 15 mL glass tubes. Air was eliminated from the tubes byusing a vacuumwater pump; tubes were then exposed to thelight in a 30 °C chamber. The results of this photosyntheticassay are shown in Fig. 2. Mutants of the N-terminallydeleted series (Fig. 2A) exhibit photosynthetic growth withthe exception of the PufXD2)26 strain. The curves in Fig. 2Ashow a lag phase varying between 15 and 50 h. Bycomparing several independent growth curves for eachmutant (data not shown), it appeared that a similar, largevariability could be observed in any strain including wild-type (compare Fig. 2A,B). Therefore the observed lag phasedid not show any correlation with the phenotype. Clearlyphotosynthetic-negative phenotypes are evidenced by thePufDX (as already reported previously [28]) and PufXD2)26curves. Also the C-terminus mutants PufX76*, PufX72*and PufX68* exhibit a nonphotosynthetic phenotype,whereas the mutant with the shortest truncation (PufX81*)

is photosynthetically competent (Fig. 2B). Surprisingly, themost extended truncation (mutant PufX54*) does not affectthe ability of photosynthetic growth.

Kinetics of cytochrome b561 reduction inducedby a single-turnover flash on ICM

The rate of electron transfer through the Qo site of cyt bc1can be measured in ICM bymonitoring the reduction of thecytochrome b561 induced by a short actinic light flash in thepresence of the inhibitor antimycin A [29]. Reduction ofthe cytochrome b561 typically shows a lag period prior to theonset of the reaction at its maximal rate. In wild-type ICM,the initial rate of this reaction, as well as the lag phase,depends on the redox state of the ubiquinone pool and onthe ubiquinone/RC stoichiometry [2,25]. For a normal sizeof ubiquinone pool (� 25 ubiquinone molecules/RC)1),upon decreasing the ambient redox potential (Eh) from 250to 100 mV at pH 7.0, the initial rate of cytochrome b561reduction increases progressively, while the lag becomesshorter. This behaviour has been attributed to the increasedavailability of prereduced ubiquinone molecules in the poolreacting at the Qo site of cyt bc1. Keeping the Eh highenough, the only ubiquinol molecule which can react at Qoand reduce the cytochrome b561 is the one released by theRC following photoexcitation, as the ubiquinone pool iscompletely preoxidized [30]. Under this condition the lagperiod is maximal, typically 1 ms in wild-type ICM. Adrastic increase of the lag phase, paralleled by a decrease inthe initial reduction rate is observed in pufX-deleted strainsas compared with wild-type [9]. Both of these effects aremaximal at Eh > 180 mV (i.e. when the ubiquinone pool isfully oxidized) and reflect a dramatic impairment in theredox interaction between the QB site of the RC and the Qosite of the cyt bc1 in the pufX-deleted strain.

We have measured the kinetics of cytochrome b561reduction of all the N- and C-terminal PufX mutants (atEh 180–220 mV) on ICM prepared from cultures grownsemi-aerobically the dark. It must be pointed out that underthese growth conditions there is no photosynthetic selectivepressure that could induce the suppression phenomenonreported in [10]. Kinetic traces recorded from the mutantsPufXD2)26andPufX54*are showninFig. 3.Thecontinuouscurves are best-fits to an exponential function; the lag dura-tionwas determinednumerically as outlined inExperimentalprocedures. The properties of the complete N-terminallydeleted and C-terminally truncated series are listed inTable 2. While the lag period of PufX54* is comparable tothatmeasured in a typical wild-type, the PufX68*, PufX72*,PufX76* as well as the PufXD2)26 exhibit an increased lagperiod usually observed in the pufX-deleted strain.

The PufXD2)4, PufXD2)7 and PufX81* show a lag periodlike that of wild-type, indicating that a short deletion at theN- and C-terminus does not affect this parameter.Measure-ments carried out onPufXD2)19 revealed an intermediate lagduration (see Table 2), making ambiguous the attribution ofthe PufXD2)19 strain to the wild-type or the PufDX cluster.

Isolation of the photosynthetic complexesfrom the mutant strains

As described previously [16], the photosynthetic complexes(PMCs) could be extracted by detergent solubilization from

Fig. 2. Growth curves of the control and mutated PufX strains under

photosynthetic conditions. The growth of the coltures was monitered by

a Klett–Summerson colorimeter. (A) Wild-type (j), PufXD2–4 (s),

PufXD2–7 (n), PufXD2–19 (,), PufDX (h); the growth curve of

PufXD2–26 (not shown in the figure for visual clarity) coincides with

that of PufDX. (B) Wild-type (j), PufX54*(s), PufX68* (n),

PufX72* (,), PufX81* (e); the growth curve of PufX 76* (not shown)

coincides with those of PufX68* and PufX72*, i.e. reveals inability of

photosynthetic growth.


themembranes and purified by centrifugation in continuoussucrose density gradients. Briefly, the final wild-type patternconsists of four bands, named PMC1, PMC2, PMC3 andPMC4 from top to bottom of the tubes (Fig. 4, tube 1).PMC1, PMC2, PMC3 and PMC4 represent LH2, LH1Ôempty ringsÕ, LH1–RC monomers and LH1–RC dimers,respectively. A consequence of the deletion of the pufX geneis the lack of LH1–RC dimer bands in the gradients (seeFig. 4A, tube 6).

Fig. 4 shows the patterns of the N-terminally deleted(Fig. 4A) and C-terminally truncated (Fig. 4B) series.PufXD2)4 and the PufX81* (the shortest deletion andtruncation, respectively) mutants exhibit the wild-type likepattern, with all four bands present. In the mutantPufXD2)7, a very faint band in the correct position ofPMC4 could be seen in the original photograph, whereas inthe other N-terminally deleted strains, PufXD2)19 andPufXD2)26 PMC4 is undetectable. Also in the mutantsPufX68*, PufX72* and PufX76* PMC4 is not seen;interestingly a fourth band not clearly separated fromPMC3, with a position intermediate between those ofPMC3 andPMC4 (between the LH1–RCmonomer and thedimer), is present in the PufX54* gradient profile (Fig. 4B,tube 2).

SDS/PAGE of the isolated LH1–RC complex

The RC : PufX stoichiometry in isolated LH1–RC com-plexes is 1 : 1 in the wild-type strain. The same unitarystoichiometry was determined in the monomeric (PMC3)and dimeric (PMC4) core complex bands [16]. The presenceof the mutated PufX can therefore be assessed in PMC3isolated from themutants. Fig. 5A shows an SDS/PAGE ofisolated PMC3 from the N-terminally deleted series. Onlythe small molecular weight region is shown. In Fig. 5A,lanes 2 and 3, corresponding to PMC3 from PufXD2)4 andPufXD2)7, respectively, above the dominant LH1 a and bbands, a band attributable to the mutated PufX is clearlyvisible. In lane 4 a faint band, attributable to PufXD2)19 isindicated by an arrow, while no band, except those of LH1a and b, can be seen in the mutant PufXD2)26 and in thePufDX strain.

Table 2. Summary of experimental data.

Strain

Light

growth

Cytochrome b561reduction lag (ms)a

Sucrose gradient-

isolated PMC

Detection of

PufX in PMC3b

Wild-type Yes 0.7 (0.2–0.8) PMC3, PMC4 Yes

PufXD2-4 Yes 1.1 (0.9–1.3) PMC3, PMC4 Yes

PufXD2-7 Yes 0.5 (0.0–1.0) PMC3 (PMC4)c Yes

PufXD2-19 Yes 3.8 (3.5–4.2) PMC3 Yes

PufXD2-26 No 5.3 (4.3–5.8) PMC3 No

PufX54* Yes 1.2 (0.5–1.4) PMC3, PMC3/4 Yes

PufX68* No 10.7 (9.5–11.1) PMC3 No

PufX72* No 6.3 (6.2–6.8) PMC3 No

PufX76* No 4.7 (4.0–5.8) PMC3 No

PufX81* Yes 0.7 (0.5–1.0) PMC3, PMC4 Yes

PufDX No 8.8 (8.8–9.1) PMC3 No

a The confidence interval within 1 SD is given in parentheses. b Detected by SDS/PAGE on sucrose gradient-isolated PMC 3. c Detectable as

a weak band in the original photograph.

Fig. 3. Cytochrome b561 reduction kinetics induced by single flash pho-

toexcitation in ICM. The continuous vertical line indicates the instant

when the actinic flash pulse was fired (time ¼ 0), the dotted vertical

line marks the beginning of the cytochrome b561 reduction at its

maximal rate. The time interval between the continuous and the dotted

vertical lines corresponds to the lag phase of the reduction kinetics. Lag

duration was evaluated by a numerical procedure as outlined under

experimental procedures. The experimental trace is represented by a

continuous line connecting the points sampled by the recording

apparatus; the best-fitting mono-exponential function is indicated by a

continuous curve. (A) ICM from strain PufXD2-26; (B) ICM from

strain PufX54*.


In Fig. 5B, data for the C-terminally truncated series areshown. The presence of PufX81* is evident in lane 2, whileno PufX band was observed in the mutants PufX76*,PufX72* and PufX68* (lanes 3, 4, 5, respectively).

A thin band very close to the LH1 a band, indicated bythe arrow, is apparent in the PufX54* mutant (lane 6).This band was excised from the gel after SDS/PAGE andthe protein was identified by using the method described inthe Experimental procedures. Briefly, the band wasdigested with 0.5 lg of the proteolytic enzyme endopro-teinase Lys-C and parts of the resulting peptide mixturewere analysed by MS (MALDI-TOF). The peptide massfingerprint obtained corresponded to fragments 17–29(TNLRLWVAFQMMK) and 5–16 (TIFNDHLNTNPK)of the PufX protein. The identity of PufX in the bandisolated was examined further by subjecting part of the

peptide mixture to separation by reversed-phase HPLC.Purified peptides were subjected to sequence analysis byEdman degradation. The sequence KTIFNDHLNTN,corresponding to the 4–14 fragment of PufX, was identi-fied.

D I S C U S S I O N

Effects of N-terminal and C-terminal PufX deletionon LH1–RC dimerization

In the LH1–PufX–RC core complex of Rh. sphaeroides, theRC : PufX stoichiometry is 1 : 1 [16]. The role of PufX as astructural organizer of the core complex has been discussedrecently in several works (rewiewed in [13]). Data on thesequence of assembly of the LH1–PufX–RC complex in vivo[31] and on protein–protein interactions between the singlepolypeptides of the complex [17] are consistent with thehypothesis that PufX interrupts the continuity of the LH1ring and switches the structure of the complex from aÔclosedÕ monomeric form to an ÔopenÕ dimeric form.Moreover, linear dicroism studies have demonstrated therole of PufX in the orientation of the RC inside the LH1

Fig. 4. Isolation of the PMCs on a sucrose gradient. The final detergent

extracts from the ICM were loaded on the top of a 10–40% sucrose

gradient and centrifuged for 19 h at 230 · 103 g. The gradient was

buffered with 50 mM Na-glycylglycine to pH 7.8, the detergents octyl-

glucoside and Na-cholate were added to the gradient at a final con-

centration of 0.6%and 0.2% (w/v), respectively. (A) Tube 1, wild-type;

tube 2, PufXD2–4; tube 3, PufXD2–7; tube 4, PufXD2–19; tube 5,

PufXD2–26; tube 6, PufDX. (B) Tube 1, wild-type; tube 2, PufX54*;

tube 3, PufX68*; tube 4, PufX72*; tube 5, PufX76*; tube 6, PufX81*.

Fig. 5. SDS/PAGE on sucrose gradient-isolated core complexes. The

proteins of the PMC3 bands isolated from the sucrose gradients (see

Fig. 4) were subjected to SDS/PAGE according to Schagger & Von

Jagow [22]. The concentrations of acrylamide and bis-acrylamide were

19.5% and 0.5% (w/v), respectively, in the separating gel and 3.9%

and 0.1% in the stacking gel. For each lane, 24 pmol PMC3, corre-

sponding to the monomeric form of the core complex, was loaded.

Only the region of low molecular mass proteins is shown in the figure.

(A) Lane 1, wild-type; lane 2, PufXD2–4; lane 3, PufXD2–7; lane 4,

PufXD2–19; lane 5, PufXD2–26; lane 6, PufDX. (B) Lane 1, wild-type;lane 2, PufX81*; lane 3, PufX76*; lane 4, PufX72*; lane 5, PufX68*;

lane 6, PufX54*; lane 7, PufDX. The position of the faint band

attributed to the PufXD2–19 protein is indicated by an arrow in lane

4 A, the position of the detected PufX54* is indicated by an arrow in

lane 6 B.


[15]. These results indicate that the PufX protein is incontact with the LH1 and the RC subunits inside the corecomplexes.

When secondary structure prediction was performed onPufX [32] the final output revealed a strong tendency tobuild ahelices at both the N- and C-termini [33] and atransmembrane a helix in the central region (Fig. 1). On thisbasis, and in view of the finding that the C-terminal part ofthe LH1 a polypeptide plays an important role in thestructure of the core complex [12], we decided to investigatethe possible structural role of the N-terminus and theC-terminus of PufX. To this aim, nine strains ofRb. sphaeroides with mutated PufX were constructed.

The dimeric form of the core complex purified from ICM[16] has been confirmed by electron microscopy [14]. Weconsider, therefore, the presence of the dimeric form(PMC4) upon isolation as an indication for dimerizationin vivo. The shortest deletion in the PufXD2)4 and theshortest truncation in PufX81* do not impair the ability ofPufX to facilitate dimerization, as a clear PMC4band can bedetected in the gradient (Fig. 4). Interestingly in the gradientof the N-terminus mutant PufXD2)7 a very faint PMC4band is visible in the original gradient photograph (unde-tectable in Fig. 4). Apparently this deletion strongly desta-bilizes the dimer to the extent that it cannot withstand fullythe membrane detergent extraction. The presence of thedimer in vivo in the mutant PufXD2)7 and presumably inPufXD2)19 is therefore not excluded. We have shownpreviously that in vitro an irreversible dissociation of thedimeric to the monomeric form of the complex from thewild-type exists: the dimer dissociates gradually intothe monomer when the octyl-glucoside concentration isincreased from 0.6 to 1.2% [16]. This result suggested thathydrophobic interactions are involved in maintaining thedimeric form. The data obtained on the PufXD2)7 andPufXD2)19 strains indicate that important protein–proteinhydrophobic interactions aremade by the PufXN-terminus.

In the case of the longest N-terminal deletion (strainPufXD2)26), the PufXD2)26 protein is not detectable in thecore complex (see below and Fig. 5A, lane 5). Correspond-ingly only the monomeric form of the complex can be seenin the gradient (Fig. 4A, tube 5).

Two main points of interest arise from the resultsobtained from the C-terminal truncation series. First, threemutants (characterized by a nonphotosynthetic phenotype),PufX76*, PufX72* and PufX68* show no dimers of theisolated core complex, whereas from the PufX54* strain afourth band, with different sedimentation characteristics onsucrose gradients, has been isolated. In the following werefer to this band, located in an intermediate positionbetween the monomer (PMC3) and the dimer (PMC4), asPMC3/4. We propose three alternative interpretations: (a)PMC3/4 represents a dimeric form in which the LH1 ringsassume a different curvature, leading to a different sedi-mentation coefficient; (b) PMC3/4 is formed by two LH1rings that lost one or two reaction centers; (c) when theC-terminal part of PufX is deleted the equilibrium betweenthe monomer and the dimer is not attained duringsedimentation.

The second interesting point is that PufX76*, PufX72*and PufX68* mutants are photosynthetically incompetent,whereas the PufX54* mutant grows photosynthetically,demonstrating that a complete removal of the C-terminus is

tolerated by the cell, while a partial truncation is photosyn-thetically lethal. The absence of PufX in PufX76*, PufX72*and PufX68* (Fig. 5B) could in principle either reflect animpairment in the insertion into the membrane of theshortened protein and/or in the assembly of PufX in theLH1–RC, or resides at transcriptional/post-translationallevel. The PufX54* protein possesses only the N-terminusand the hydrophobic transmembrane helix, whereas theother mutants have in addition part of the C-terminus. Wesuggest that the presence of a partial C-terminus leads to amisfolding that impedes the insertion/assembly of PufX inthe membrane complex.

Parkes-Loach et al. [34] have recently reported thatmature forms of PufX extracted from cells of Rb. sphaer-oides and Rb. capsulatus contains 12 and nine fewer aminoacids, respectively, at the C-terminal end of the protein thanare encoded by their pufX genes. These data are inconsistentwith our previous report [16], where a PufX with aC-terminal six-histidine tail has been used to determinethe RC : PufX stoichiometry by Western blot analysis withanti-His6 antibodies. However the genetic background ofthe strains used is different: in our studies (present paper and[16]) both the LH2 and the LH1 antenna systems arepresent, while in the work of Parkes-Loach et al. an LH2–,LH1– strain and an LH2– strain from Rb. sphaeroides andfrom Rb. capsulatus, respectively, have been used to extractPufX. We can suppose that the discrepancy is related to thepresence of the LH2 which could influence the shorteningprocesses of the assembled PufX protein.

The exchange of ubiquinone between the RCand the cyt bc1 in the presence of mutatedPufX protein

The role of the PufX protein in facilitating the ubiquinone/ubiquinol exchange between the QB site of the RC and theubiquinone pool has been demonstrated in Rb. sphaeroideswild-type strains [7,8]. It has been proposed that PufXfacilitates ubiquinone exchange by determining the struc-tural supramolecular organization of the LH1–PufX–RCcomplex [12].

In this work, PufX has been detected by SDS/PAGE incore complexes (Fig. 5) isolated from the N-terminusmutants PufXD2)4, PufXD2)7, PufXD2)19 and from theC-terminus mutants PufX54*, PufX81*. The evidence thatthese are the only mutants which are photosyntheticallycompetent (see Table 2) is in accordance with previousresults on the requirement of the PufX protein forphotosynthetic growth and suggests that the assembly ofthe wild-type or mutated PufX protein in the core complexis necessary for efficient light energy transduction. In theother mutants examined, PufXD2)26, PufX68*, PufX72*and PufX76* no PufX protein could be detected on SDS/PAGE after isolation of the complex.

Assaying on ICM the reduction kinetics of the cyto-chrome b561 induced by a single actinic flash in the mutantsPufXD2)4, PufXD2)7, PufX54* and PufX81*, we found alag time between the flash excitation and the onset ofcytochrome b561 reduction close to that observed in wild-type ICM. This is indicative of a fast ubiquinone exchangebetween the reaction center QB site and the cyt bc1 Qo site.In the case of the shortest N-terminal deletion andC-terminal truncation (PufXD2-4 and PufX81*, respectively)


this result was expected; in these two mutants a dimericform of the core complex could be isolated. On the contrary,we obtained evidence of a less stable dimer in mutantPufXD2)7 and observed a band intermediate between thatof the monomeric and the dimeric form (see above) inmutant PufX54*. As in these last two mutants a short lagwas observed (see Table 2), apparently the presence of astable dimer is not a necessary requisite for a fast RC/bc1redox interaction, which is associated with a photosyntheticphenotype.

As an alternative explanation the monomeric and thedimeric form of the LH1–PufX–RC could both be presentin vivo; in the presence of an intact PufX the dimeric formwould prevail, while altered equilibria arising from muta-tions on PufX could affect the stationary concentration ofthe dimer in the membranes.

In the PufXD2)19 strain the dimeric form is even moredestabilized, as no PMC4 can be isolated. Measurements ofthe lag in cytochrome b561 reduction in ICM from thismutant yielded values intermediate between those usuallyobtained in the wild-type and in the PufDX strain, withsome variability between preparations from different cul-tures. Considering that the same amount of LH1–RC hasbeen loaded in all lanes of the SDS/PAGE gel in Fig. 5A,the weaker intensity of the PufXD2)19 band (lane4) suggeststhat the amount of PufX per LH1–RC complex is lower inthis mutant. Therefore it is likely that a mixture ofmonomeric LH1–RC with and without PufXD2)19 isisolated on the sucrose gradient. The occurrence of a mixedpopulation of LH1–RC core complex in the ICM wouldexplain the variability in the duration of the lag ofcytochrome b561 reduction kinetics. The presence of thePufXD2)26 within the isolated core complex cannot beexcluded in the SDS/PAGE shown in Fig. 5A, due to apossible overlapping with the a subunit of the LH1complex. However a significant efficiency of PufXD2)26insertion in the core complex seems unlikely, due to thenonphotosynthetic phenotype of this strain and to thepronunced lag in the cytochrome b561 reduction kinetics (seeTable 2), systematically found in chromatophores from thepufX-deleted strain.

Organization of the Q-cycle complexes

The dimeric organization of the LH1–PufX–RC has beendemonstrated directly in the membranes of Rb. sphaeroidesby electron microscopy [14]. In this paper, the authorstentatively attribute a positive electrondense region in thetwo-dimensional projection of the dimer to cyt bc1 andinterpret the S-shaped structure of the projection map as asupercomplex formed by the LH1–RC and cyt bc1 in a 2 : 1stoichiometry.

Some considerations on this point can be made in thelight of our results. The presence in vitro of a less stabledimer in the mutant PufXD2)7 neither affects the photo-synthetic capability of the bacteria nor the efficiency ofexchange of the ubiquinol molecules between the RC andthe bc1, as judged from the reduction kinetics of cyto-chrome b561 measured in ICM. Also the mutant PufD2)19,in which the dimeric form cannot be detected in the isolatedcore complex, exhibits a photosynthetic phenotype. In thesetwo mutants, the photosynthetic phenotype suggests thepresence in vivo of an open monomeric complex (or a

prevalence of it with respect to the wild-type situation),consisting of an incomplete single LH1 ring containing oneRC.

The photosynthetic ability in PufX54* is consistent withthe fast RC/bc1 ubiquinol exchange observed in ICM; onthe other hand, the structural organization of the corecomplexes and/or the possible monomer–dimer equilibriumseem to be appreciably perturbed also in this mutant asjudged from the different position of the PMC3/4 band(Fig. 4B) after isolation of the photosynthetic complexes onlinear sucrose gradient. It is possible that the dimer form isnot required as long as a reorganized core complex canefficiently shuttle quinones between the RC and the bc1complex. The PMC3/4 isolated complex stimulates ourinterest, and further studies are in progress to understandthe nature of the PufX54* mutant.

In conclusion, our data indicate that both the N- andC-terminal portions of the PufX protein play a complex rolein organizing the structure of the LH1–RC complex; theN-terminal region would be responsible mainly for theformation of a stable dimer, whereas the C-terminal portionwould be involved mainly in PufX insertion/assembly. Thetransmembrane helix region of PufX appears to be sufficientto allow a fast quinone exchange between the core and thecytochrome b561 complex. Interestingly this conclusion fitswell with the recent work of Parkes-Loach et al. [34],showing that the interaction between the hydrophobic PufXregion and the LH1-a polypeptide has an inhibitory effecton the formation of the LH1 complex. This result suggeststhat the central core of the PufX protein is responsible of thebreak in the continuity of the LH1 ring in vivo [14], allowinga faster diffusion of the quinone molecules from/toward theRC QB site.

A C K N O W L E D G E M E N T S

We thank B. A. Melandri and P. Turina for fruitful discussions,

C. Weyrauch, U. Schimanko and N. Mele for technical assistance.

F. F. and J. W. were recipients ofM.P.I. postdoctoral fellowships. This

work was supported by grant PRIN/99, Bioenergetica e trasporto di

membrana from the Italian MURST and from The Fonds der

Chemischen Industrie.

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Documents

Role of the N- and C-terminal regions of the PufX protein in the structural organization of the photosynthetic core complex of Rhodobacter sphaeroides