Indian Journal of Biochemistry & Biophysics Vol. 37 , December 2000, pp. 351-359

), (- Minireview ( -

352 INDIAN J BIOCHEM BIOPHYS, VOL. 37, DECEMBER 2000

Table I - Subuni ts o f pholosystem I [TM : transmembrane protein, with number of transmembrane helices : S : stromal protein ; L: lumenal protein]

Subunit name Di stribution Locali zation Function

PsaA , PsaB cyanobacteri a, chloroplasts TM (each I I) Light harvesting, charge separation PsaC cyanobacteri a, chloroplasts S Electron acceptor PsaD cyanobacteri a, chloroplasts S Binding of ferredoxinlflavodoxin PsaE cyanobacteri a. ch loroplasts S Binding of ferredoxinlflavodoxin PsaF cyanobacte ri a, chloroplasts TM (2) Binding of plastocyaninlcytochrome C6 FsaG chloroplas ts TM ( 1-2) Interaction with LHC I PsaH chloroplasts S Psal cyanobacteria, chloroplasts TM ( I) PsaJ cyanobacteria, ch loroplasts TM ( I) PsaK cyanobacteria, chloroplasts TM (2) PsaL cyanobacteria, chloroplasts TM (2) PsaM cyanobac teria, chloroplasts TM ( I) PsaN chl oropl asts L

chloroplasts, have already been sequenced: The unicellular cyanobacterium Synechocystis PCC 6803 was sequenced in 1994 (ref. 5,6), the heterocyst forming cyanobacterium Anabael/a was completed by the same group (http://www.kazusa.orjp/cyano/ anabaena!) only in year 2000. Sequencing projects for a lgae like Chlamydomonas (http://www.botany.duke. eduichlamy/ChlamyGen/genome.htm) and for higher plants like Arabidopsis thaliana (http://synteny.nott. ac .uk/agr/agr.html), rice (http://www.dna.affrc.gojp:82J) and maize (http://www.zmdb.iastate.edu or http:// www.agron .missouri.edu/) are under way and will be completed soon. In addition. chloroplast genomes from different species have been known for almost a decade. These data allow a reverse genetics approach [ 0 determine the function of unknown proteins . Some of the newly discovered biogenesi s proteins were identified by such an approach .

What else can we learn from sequence data concerning PS I? Genes for PS 1 subunits are distributed all over the cyanobacterial genome with psaA and psaB, psaJ and psaF, and psal and psaL cotranscribed in Synechocystis7-9. In algae and higher plants the situation is more complicated as only some proteins are chloroplast encoded (PsaA, PsaB, PsaC, !Psal, PsaJ) while the rest is nuclear encoded 'o. But even in this case the psaA and psa8 genes are cotranscribed " . The alga Chlalllydolllonas reinhardtii is widely used as a model system for higher plant photosynthesis and represents an even more complicated picture : psaA and psa8 are transcribed independently and psaA is made up from three widely separated exons 12.13.

Obvious questions emerge from the genome organization described above: How is the expression

Interact ion with LHC I ? Functional organization of PsaL Functional organization of PsaF ?, Interaction with LHC I Trimerization Cyclic electron flow ?

of all these genes regulated in a way that different subunits are synthesized in the right stoichiometry? How is gene expression coordinated between the nucleus and the chloroplast in eukaryotic cells? How is PsaA synthesized in Chlamydomonas and why is this process so complicated?

Unfortunately, we can only partially answer these questions. Most data are derived from targeted deletions of individual genes followed by analysis of isolated PS I complexes from these mutants. The level of PsaA and PsaB seems to control the accumulation of all other subunits . Deletion of e ither psaA or psa8 lead to a loss of other PS I subunits in thylakoid membranes of Chlalllydomonas and cyanobacteria7. 14.15. Site-directed mutagenesis of these subunits often results in reduced or no accumulation of PS I, which further emphasizes the central role of these subunits l6 . Deletion of psaC, the only other PS 1 gene that binds an important cofactor, leads to the accumulation of a PsaD and PsaE deficient PS I complex in cyano-bacteria 17,18. The same deletion in Chlamydomonas leads to a total loss of PS J subunits, which highl ights the general tendency of Chlamydomonas to rapid ly degrade partially assembled membrane protein complexes l9 .

For the special case of psaA trans-splicing in Chlamydomonas, several nuclear factors have been found through mutagenesis screening. These fall into three classes 13.20.21: No splicing at all, exon 1 .. 2 splicing defective and exon 2-3 splicing defec ti ve. In total, 14 different factors have been fou nd; taking into account that the screen was not saturated more factors may as yet be undetected . In most cases the molec ul ar identities of these factors are not known. ncA. a chloroplast gene expressing an RNA which is

SCHWABE & KRUIP: BIOGENESIS AND ASSEMBLY OF PHOTOSYSTEM I 353

necessary for exon 1-2 splicing, is the only identified I·· f ~? sp ICI I' ;:'; actor up to now--. Inactivation of other subunits in cyanobacteria

shows only limited effects on the presence of other proteins within the PS I complexes. For example, deletion of psaD, psa£ and psaK leads to assembled PS I complexes, which in case of the psaD deletion mutant are more fragile but still contain all other subunits23.27. Although psaL deletion prevents trimerization, it sti ll results in functionally intact monomeric PS 128.29 . Deletion of psa/ or of psaJ results in a reduced level of PsaL or PsaF, respectivel/,3o.

In all these cases however, the question whether gene deletion affects the assembly of other subunits due to decreased synthesis (e.g. through transcriptional or translational control) or increased proteolysis (posttranslational control) has not been ana lyzed and remains to be solved. In addition, neither specific transcription factors nor regulators are known (with the exception of splic ing factors in case of Chlamydomonas) and we can only speculate that the mRNA may be directed by proteins to specific membrane sites where protein synthesis and assembly of the PS I complex takes place.

Assembly of PS I The sequence of subunit assembly to yield

functional PS I has been already discussed in the previous chapter. In short, the heterodimer PsaA and PsaB seems to be the folding core on which the other subunits are assembled. Assembly of PsaL is dependent upon the presence of PsaI and assembly of PsaF upon the presence of PsaJ9•30. If PsaC is absent, no PsaO and PsaE become assembled into PS 117.18. In addition, mutants in which the essential cysteine

ligands of the f4Fe4S] clusters FA or FB were changed to alanine, could not grow photoautotmphically. No assembled PsaC nor any PsaO or PsaE could be detected in vivo in these PS 1 complexes31. This implies that the incorporation of two [4Fe4S] clusters into PsaC is essential for its assembly. Golbeck and coworkers deve loped an ill vitro system to rebind the stromal subunit PsaC to PsaC, -0 and -E defic ient PS 132. Thi s turned out to be possible only under reducing, anaerobic conditions in the presence of FeCI3 and Na2S, Under these conditions Golbeck ' s group was able to detect assembled PsaC, but the PS I complexes were highly susceptible towards oxygen 32. However, if reconstitution was carried out together with PsaO and PsaE, stable incorporation of PsaC

could be shown33. Further, mutations that lead to destruction of only one PsaC iron-sulfur center failed to assemble PsaC in an ill vitro re-binding assa/4 . Taken together, all data show, that (1) assembly of PsaC, -0 and -E can occur without the he lp of other proteins and that (2) correct formation of both PsaC iron-sulfur centers is a prerequisite for assembly of PsaC, -0 and -E. However, we can assume that additional proteins for iron-sulfur cluster incorporation exist in vivo (see below).

The assembly process of the other two stromal subunits PsaD and PsaE was also addressed experimentally. Radiolabeled PsaE could be incorporated into PsaE deficient PS I complexes in vitro35 . Thus PsaE contains sufficient information for its own assembly. However, the stability of the assembled PsaE depends on the presence of other subunits like PsaD and PsaJ, _F35. Similarly, radiolabled PsaD could be incorporated into PsaD deficient PS I complexes36. Later it was shown that PsaD assembly in chloroplasts is a two-step process where a PsaD precursor is first incorporated into PS 1 and then processed to the mature form37.

Recently new proteins were identified which are no structural part of PS I but which apparently are necessary for some steps in PS I biogenesis (Table 2). Only for one protein , RubA, a likely role in PS I assembly can be assigned. The functions of the other proteins, Ycf3 , Ycf4, BtpA and FtsH are as yet unknown . All show the same phenotype after deletion of their corresponding genes: a severe reduction of PS I content.

When the rubA gene was deleted by Don Bryant's group in Synechococcus PCC 7002, the cells could not grow photoautotrophically. Subsequent anal ysis traced the defect to inactive PS I while the PS II complex was functional. Isolated PS 1 complexes from the mutant had not only lost the stromal subunits PsaC, -D and -E but also the [4Fe4S] cluster F/8. The rubA gene codes for a small rubredoxin-like protein , which contains a C-terminal transmembrane helix . RubA seems to be essential for the generation of the three iron-sulfur clusters found in PS I. This may be the first identified protein involved in cofactor biogenesis/integration of PS I. It shows as well that iron-sulfur cluster biogenesis is a posttranslati onal process as small amounts of PsaC, PsaD and PsaE can st ill be detec ted within the RubA defici en t cells38. Recently , NifS-like protei ns, which are in volved in iron-sulfur cluster biogenes is by removing sulfur from free cysteine, have received special attention. The

354 INDIAN 1 BIOCHEM BIOPHYS. VOL. 37. DECEMBER 2000

Table 2 - Li st of proteins. which are presumably involved in PS I biogenesis

Protein name Organi sm Prote in features Effect Literature

RubA Cyanobacteria Rubredoxin-like protein; PS I iron-sul fur centers Shen el al .• 1998 around 50 kDa not assembled

Product of orf Cyanobacteria NifS -like protein. 42 kDa Involved in PsaC iron- Anto nkine and Golbeck , sll 0704 sulfur cluster asse mbl y pers. communi cation Ycf3 Cyanobacteria. Contains three TPR PS I not detectable Boudreau el al. , 1997 ;

chloroplasts motifs ; membrane Ruf el al., 1997; associa ted 20 kDa protein Kruip e/ al ., unpubli shed

results Ycf4 Cyanobacteria. Very bas ic, 20 kDa PS I amount reduced or Boudreau el aI., 1997;

chloroplas ts protein, membrane not detectable Wilde el al. , 1995 associated Kruip el al. , unpubli shed

results BtpA Cyanobacteria Membrane associated PS I amollnt reduced Bartsevich and Pakrasi,

30 kDa protein 1997 FtsH Cyanobacteria Around 68 kDa PS I amount reduced , Mann , personal

me mbrane prote in, AAA PsaE missing commu nication ~rotease

genome of SYlleehoeystis conta in s three open-reading frames for NifS-like proteins whose biological roles were unknown. In vitro studies have already shown that the gene product of orf slr0387 can catalyze iron-

If . . . f d . 39 SU . ur-center tI1corporatton 111 to erre oXln . Moreover, another NifS-like protein, encoded by s ll0704, is able to incorporate both iron-sulfur centers into PsaC (M.L. Antonkine and J.H. Golbeck, personal communication). Thus at least two additional proteins seem to be necessary fo r iron-sulfur cluster assembly in PS I.

Deletion of the conserved open-reading frame yef3 in Chlamydoll1onas by John-Dav id Rochaix 's group resulted in a complete loss of functional PS 140, whereas PS II was unaffected . Similarly, de letion of the tobacco homologue4 1 or the cyanobacterial homologue (K. Gloddek and J. Kruip, unpubli shed data) resulted in the same PS I less phenotype. Almost all PS I subunits were absent or barely detectable by antibodies in these mutants (J. Kruip , unpublished

I )40 4 1 A . h . . . b resu ts ' . s ne lt er transcnptlOn nor n osome loading of PS I genes psaA and psaB are affected, Y cD seems to ac t posttranslationally in PS I biogenesis40.41 . The yef3 gene is highly conserved from cyanobacteria to higher plants (around 70-80% identity) and codes for a 20 kDa protein . After fractionation studies the Y cD protein was found associated with thylakoid membranes in Chlamydomonas but could be removed from these memb;anes by treatment with chaotrophs4o. Immuno-e lectron microscopy using gold-labeled antibodies showed exc lusi ve labe ling of thylakoid membranes in the cyanobacterium Syneehoeystis (D. Kahmann , J. Kruip , unpublished data) and treatment with

chaotrophs resulted in membrane re lease of Y cf3 (M . Masgaj , J. Kruip, unpublished data). It can be concluded that Y cf3 is a membrane-associated protein . According to sequence comparisons Y cD contains three so-called TPR motifs4o. This motif consists of a degenerate 34 ami no acid stretch42,43 and TPR-containing proteins are usuall y in volved in multi protein complex formation important for the function of chaperone, cell cycle, transcription and protein-transport complexes43. Protein phosphatase 5, a protein containing three TPR motifs could be crystallized44 and it was shown that the TPR motif folds into two helices , which are tightl y pac ked aga inst each other. Das et al. proposed that tandem TPR motifs could build up a right-handed super-heli x with a helical repeat of approximately seven TPR motifs44• The cavity within the heli x would be 600 nm deep and 420 nm wide and could accommodate an u-helix of its potential binding partner. Gelfiltration data of recombinant Ycf3 point to the existence of an oligomeric complex containing more than eight Ycf3 molecules (K. Gloddek, J.Kruip, unpublished results). Thus it is highly likely that Ycf3 interacts with another, yet to be characterized protein. In tobacco and mustard the yef3 gene is located upstream of the psaA/psaB genes and may be cotranscribed wi th psaA41 ,45 . The possibility that Ycf3 may interac t direc tly with PsaA awaits experimental confirmation.

The same approach led to the deletion of the conserved open-reading frame yef4 in Chlamydomonas reinhardtii by John-David Rochaix ' s group and resulted like the yef3 knock-out in a complete loss of almost all PS I subunits40. As for Ycf3 , it was shown that Y cf4 loss does not influence PS I gene

SCHWABE & KRUIP: BIOGENESIS AND ASSEMBLY OF PHOTOSYSTEM I 355

transcription or ribosome loading, therefore pointing to a posttranslational PS I biogenesis process in which Ycf4 is involved. In contrast, when yef4 was deleted earlier in Syneehoeystis, only a shift in the PS I to PS II stoichiometry rather than a complete loss of PS I was observed: PS I levels decreased nearly 50 % in relation to PS 1146. Nevertheless the remaining PS I complexes were correctly assembled and functionally intact (1. Kruip, A. Wilde and T. Borner, unpublished data). This highlights that although cyanobacteria can serve as model systems for chloroplast and higher plants, it should be kept in mind that important differences occur. There might be an alternative pathway of PS I biogenesis in cyanobacteria which does not depend on Ycf4. Furthermore, in Chlamydomonas the yef3 and yef4 genes are co-transcribed40 whereas they are independently expressed in Syneehoeystis. Ycf4 is not as highly conserved (40-50 % sequence identity) between cyanobacteria and higher plants as Y cf340. The gene codes for a 20 kDa protein which, according to hydropathy analysis, may have two N-terminal transmembrane helices40.46. Fractionation studies in Chlamydomonas showed a cofractionation with thylakoid membranes from which it could be removed by sodium carbonate treatment40. Direct localization using gold-labeled antibodies also showed a thylakoid membrane association of Y cf4 in Syneehoeystis (u. Kahmann, 1. Kruip, unpublished data). In this case, Y cf4 could be released from membranes by urea treatment. These data are compatible with a thylakoid membrane-associated protein despite the presence of two hydrophobic stretches, which may form transmembrane helices. At present, we can not rule out the possibility that Y cf4 contains transmembrane helices but still can be released from membranes under certain conditions as such behavior was shown before in the case of cytochrome b6f subunits

47.

A random screen for photosynthesis deficient mutants in the cyanobacterium Synechocystis pce 6803 lead to the discovery of the BtpA protein48. The mutant has a missense mutation of the btpA gene leading to an 85-90% decrease of PS I on a protein level. The btpA gene codes for a 30 kDa protein , which may have two transmembrane helices according to hydropathy plots. As the rnRNA levels of psaAlpsaB are unchanged in the mutant, a translationallposttranslational role for BtpA in PS I biogenesis was inferred48. Fractionation studies localized the protein in thylakoid membranes; further phase partitioning experiments concluded it to be a

membrane associated protein residing on the cytoplasmic face of the membrane49. Direct visualization of BtpA localization by gold-coupled antibodies further confirms this localization (u. Kahmann, 1. Kruip, unpublished data). The fact that close relatives of the BtpA protein can also be found in archaebacteria like Methanococcus jannaschii and in eukaryotes like Caenorhabditis elegans clearly shows that despite a role in photosystem I, biogenesis BtpA must be involved in some general kind of process which is important for all life forms.

The complete genome of Synechocystis contains four genes homologous to the E. coli fisH gene. In E. coli this membrane bound ATPase functions either in a proteolytic mode or in a chaperone modeso. For example, in the proteolytic mode FtsH degrades unassembled membrane proteins rapidly as was shown for the ATPase subunit as' . Deletion of one of the four open reading frames (slr0228) results in a 60% decrease of PS I levels. Further, the assembled and functionally intact PS I complexes show altered fluorescence emission characteristics and a specific loss of the PsaE subunit (N. H. Mann, personal communication). Whether FtsH is indeed important for the biogenesis of a specific PS I feature still has to be proved. For the cytochrome bc, complex however, it was already shown that a protein containing an AAA motif (Bcslp) is necessary for integration of the Rieske and the QcrlOp subunitS2. These authors speculated that Bcslp might function as a molecular chaperone for membrane protein assembly.

Almost no information about integration of cofactors other than the iron-sulfur clusters is available. Cofactor loading could be carried out co- or posttranslationally. In case of chlorophyll some data suggest that it is integrated cotranslationally: Translation of the psbA gene (coding for the PS II reaction center protein Dl) depends on the availability of chlorophyll (chi), if chi is absent translation will be arresteds3. However, this effect could not be reproduced in a cell free systemS4. Analysis of psbA translation in a PS I less strain with re'duced chlorophyll synthesis capabilities clearly showed that chlorophyll availability controls psbA transcription levels in vivoss. We do not have experimental data to draw similar conclusions for PS I proteins. This also holds true for the integration of carotenoids, phylloquinones. The involvement of other proteins in these steps is to be expected .

Dynamics of PS I Once functiona l PS I has assembled 111 the

356 INDIAN J BIOCHEM BIOPHYS, VOL. 37, DECEMBER 2000

thylakoid membrane it can interact dynamically with other membrane protein complexes. In cyanobacteria, monomeric PS I can interact with each other to form stable trimeric complexes . In addition , a highly regulated dynamic interaction with phycobilisomes (PBS) can occur under certain physiological conditions (see review56.57). Plants do not contain PBS but hydrophobic light harvesting molecules (LHC I), which bind additional chI's (see review58.88). They form specific supercomplexes with monomeric PS I and thereby increase the absorption coefficient for photons.

We will first discuss cyanobacterial PS I. Since first reports about the existence of trimeric PS I in cyanobacteria were published, a controversy deve-loped59. Either the trimeric form was regarded simply as a non-physiological artifact induced by the d d . . I . 6061 h . . etergent treatment unng ISO atlon ' or t e tnmenc form was seen as physiologically important and active form (for example62,63). In the meantime sufficient data have accumulated to say with confidence that the trimeric form is indeed the physiological important one: Although in most cyanobacteria monomeric and trimeric PS I do not show any significant differences in various biophysical assays64.65, it was possible to show the presence of particles with dimensions of tri meric PS I in thylakoid membranes64.66. Tsiotis el ai . raised a monoclonal antibody that recognizes an epitope, which is present only in trimers67. This antibody reacts also with native thylakoid membranes , thus proving the existence of trimers within these membranes67. For SYllechocyslis, an equilibrium between monomers and trimers exists in membranes, which can be influenced in vitro by the ionic strength of the medium64 . From the cyanobacterium Spirt/lina piatell sis PS I can also be isolated in a monomeric and a trimeric form but in this organism the oligomeric states differ in their fluorescence behavior: In 77 K fl uorescence emission spectra, monomeric PS I shows a band at 720-725 nm whereas trimeric PS I shows an additional emission band between 750 and 760 nm68. Thus the extreme red fluorescence emission of trimeric PS I can be used as an internal marker for the existence of trimeric PS I within intact cells. Indeed it could be observed that the majority of PS I within Spirulilla cells occurs in the trimeric form69.

Which structural determinants are responsible for the specific trimerization process? After deletion of psaL only monomers can be isolated from Synechocystis making this subunit a prime candidate for the trimerization site28,29,70. The structure

elucidation confirmed these findings and showed, albe;t not at molecular resolution , a specific trimerization domain made up from the PsaL subunit3.4. An in vitro reconstitution system for trimerization of Spirulina PS I was described which showed that the stromal subunits , most probably PsaD since it can be crosslinked to PsaL63, are also involved in the trimerization process71 . Recently, another important factor for trimerization could be found which may shed some light on the physiological significance of the monomer-trimer equilibrium. Calcium in the micromolar range was found to be essential for the trimerization, only by strontium could it be partially replaced (1. Kruip , V.P. Chitnis, P.R. Chitnis, unpublished results). Furthermore, it could be shown that calcium binds specifically to the PsaL protein (V.P. Chitnis, P.R. Chitnis, personal communication). PsaL from unicellular cyanobacteria contains 1-2 conserved acidic residues in the first lumenal loop, which are absent in higher plants. These residues are likely candidates for calcium binding sites and site-directed mutagenesis to prove this prediction is under way. From these data the following worlUng model can be built: The normal oligomeric state of PS I in cyanobacteria is the trimeric form. This form can connect to phycobilisomes, drastically increasing its absorption coefficient5664.72. If a certain ~pH is generated and pH in the lumen falls under a certain threshold (pH < 6), calcium is released from its binding sides due to the acidic residues being protonated . This leads to monomerization and the monomers are not able to connect efficiently to the phycobili somes. The photosynthetic electron transport decreases until the ~pH can be used. This mechanism may adjust the photosynthetic electron transport rate to the actual demand. However, mutants of Synechococcus PCC 7002 with deleted psaL and psal genes still show state transitions, they are only three times faster than for the wild type70. PsaL and a trimeric PS I is thus not strictly necessary for phycobilisome binding. Nevertheless, the facts that (1) smaller size of monomeric PS I results in faster state transitions and that (2) membrane fluidity effects state transitions 73 show the importance of dynamic membrane protein movement for cell physiology.

In case of higher plants only monomeric PS I can be found, although the PsaL subunit is present. Trimerization may be prevented in this case by the psaH subunit which is a unique feature of chloroplast PS I. Crosslinking experiments have located the PsaH

SCHWABE & KRUIP: BIOGENESIS AND ASSEMBLY OF PHOTOSYSTEM I 357

subunit near PsaL74 and there it may prevent trimerization simply by sterical hindrance. Instead of water-soluble PBS, eukaryotic cells contain chl-binding membrane proteins (LHC I), which serve as external antenna for PS 158. Older work suggested that higher plant PS I is surrounded by a ring of eight LHC I molecules which serve as external antenna75• A re-investigation using a larger data set draws a slightly different conclusion: PS I is surrounded unevenly by 4-6 LHC I molecules76 . This is concurrent ~ith crosslinking data in which only the PsaG and -K subunits could be crosslinked to LHC I molecules74. Boekema et at. favour a location of LHC I on the side of the PsaF protein 76. Such a location would explain results from Hippler et al. that a PsaF defecti ve Chlamydomonas strain which is high light sensitive can be rescued by mutations which prevent correct LHC assembly77. This also highlights the importance of dynami c interactions between PS I and LHC for ce ll viabi lity. The dynamic in vivo situation within biologica l membranes will be a research topic with high priority in the future.

Unresolved questions and future research topics We have not yet touched on the last step in the ' life

cycle ' of PS I, its degradation . One would expect a specific, highl y regulated degradation pathway for PS I. Unfortunate ly this question is difficult to address experimentall y, since PS I is a very stable protein complex with a long half-life time. Until now no proteases or factors are known which might be in volved in PS I degradation . Herrmann and colleagues identified several proteolytic activities inside chloroplasts and started a detailed biochemical and molecular biology characterization of these78.79 . Thi s approach could lead to the discovery of a proteol ytic system for PS 1.

In contrast to PS I, much work has already been done to unravel PS II degradation, which undergoes a rapid turnover under high li ght conditions8o• Despite this effort no PS II specific protease has been found until now. Recently, it could be shown that PS II degradation is GTP dependent and that FtsH might be involvedsi. This findin g opens up the possibility that the general membrane protease FtsH is al so involved in PS I degradati on.

Exciting di scoveries have a lready been made for the biogenesis of another photosynthetic membrane protein, the light-harvest ing complex . Its membrane targeting occurs cotranslationall y, is GTP dependent and needs the presence of FtsY, the chloroplast

homologue of the bacterial signal recognition particle82. For the final membrane integration of LHC the membrane protein albin03 was found to be absolutely necessar/3. It is reasonable to assume that such proteins will as well participate in PS I biogenesis .

New experimental approaches should lead to faster characterization of unknown proteins and their biological function . In particular transcriptomics84, proteomics85.86 and large scale screening of protein interactions with the yeast two-hybrid system87 will speed up elucidating open questions in membrane protein assembly.

Acknowledgement We wish to thank Dr. M . R6gner for his continuous

support and many colleagues for stimulating discussions and sharing results prior to publication, particularly Dr. P. R. Chitnis, Dr. N. Karapetyan, Dr. M . Hippler, Dr. N. Mann and Dr. Y. Takahashi . Research in the author' s lab was supported by the DFG (SFB 480, project C2), Fonds der Chemi schen Industrie and through a NATO grant (awarded to Dr. N. Karat~yan , M . R6gner and J. Kruip) .

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