Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M406955200 on August 16, 2004

J. Biol. Chem., Vol. 279, Issue 43, 44621-44627, October 22, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/43/44621    most recent
M406955200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by de Vitry, C.
Right arrow Articles by Kallas, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Vitry, C.
Right arrow Articles by Kallas, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Chloroplast Rieske Iron-Sulfur Protein

AT THE CROSSROAD OF ELECTRON TRANSPORT AND SIGNAL TRANSDUCTION*

Catherine de Vitry{ddagger}§, Yexin Ouyang¶, Giovanni Finazzi{ddagger}, Francis-André Wollman{ddagger}, and Toivo Kallas¶

From the {ddagger}Physiologie Membranaire et Moléculaire du Chloroplaste CNRS UPR 1261, Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France and Department of Biology and Microbiology, University of Wisconsin, Oshkosh, Wisconsin 54901

Received for publication, June 22, 2004 , and in revised form, August 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have addressed the functional and structural roles of three domains of the chloroplast Rieske iron-sulfur protein; that is, the flexible hinge that connects the transmembrane helix to the soluble cluster-bearing domain, the N-terminal stromal protruding domain, and the transmembrane helix. To this aim mutants were generated in the green alga Chlamydomonas reinhardtii. Their capacities to assemble the cytochrome b f complex, perform plastoquinol oxidation, and signal 6redox-induced activation of the light-harvesting complex II kinase during state transition were tested in vivo. Deletion of one residue and extensions of up to five residues in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency. Deletion of three residues ({Delta}3G) dramatically decreased reaction rates by a factor of ~10. These data indicate that the chloroplast iron-sulfur protein-linking domain is much more flexible than that of its counterpart in mitochondria. Despite greatly slowed catalysis in the {Delta}3G mutant, there was no apparent delay in light-harvesting complex II kinase activation or state transitions. This indicates that conformational changes occurring in the Rieske protein did not represent a limiting step for kinase activation within the time scale tested. No phenotype could be associated with mutations in the N-terminal stromal-exposed domain. In contrast, the N17V mutation in the Rieske protein transmembrane helix resulted in a large decrease in the cytochrome f synthesis rate. This reveals that the Rieske protein transmembrane helix plays an active role in assembly-mediated control of cytochrome f synthesis. We propose a structural model to interpret this phenomenon based on the C. reinhardtii cytochrome b6f structure.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytochrome (cyt)1 b6f complex couples electron and proton transfer in photosynthetic organisms. It catalyzes the oxidation of plastoquinol (PQH2) and the reduction of plastocyanin. The complex contains four major subunits: cyt f, cyt b6, Rieske protein, and subunit IV (for review, see Ref. 1). Four of its redox cofactors have long been identified; they are a c-type heme in cyt f, two b-type hemes in cyt b6 (bL and bH), and an Fe2S2 cluster in the Rieske iron-sulfur protein (ISP). A new heme ci (or x) bound to cyt b6 close to the quinone reduction Qi site, a chlorophyll, a carotenoid, and specific lipids have also been identified in the cyt b6f structures from the green alga Chlamydomonas reinhardtii (Chlamydomonas) (2) and the cyanobacterium, Mastigocladus laminosus (3). Additional biochemical and spectroscopic characterization of heme ci has been obtained recently (4).

The ISP is essential for PQH2 docking and for diverting its electrons into the "high" and "low potential pathways." After PQH2 oxidation at the Qo site located close to the lumenal face of the thylakoid membrane, one electron is injected in the "high potential chain" consisting of the ISP, cyt f, and plastocyanin. This chain channels electrons from photosystem (PS) II to PS I. The second electron from PQH2 is injected into the "low potential chain" comprised of the bL, bH, and likely the ci hemes. This second pathway results in electron transfer across the membrane to the Qi site, located close to the stromal face of the thylakoid membrane. According to the Q cycle mechanism (5, 6), the genesis of a PQH2 at the Qi site increases the H+/e– stoichiometry of electron flow. Diversion of electrons to the high and low potential branches is mediated by an ISP conformational change. In the related mitochondrial bc1 cyt complexes, the rotational swing of the ISP extramembrane domain leads to a cluster movement over 20 Å, which prevents a double electron injection into the high potential chain (for review, see Ref. 1). Evidence for this movement was provided by three-dimensional cyt bc1 structures (7–9), electron paramagnetic resonance spectroscopy (10), and mutagenesis studies (for review, see Ref. 11). Several lines of evidence support an ISP domain movement in the b6f complex; they are two-dimensional crystals (12), viscosity studies (13), electron transfer from ISP to cyt f in vitro (14), inhibitor binding (15), mutagenesis studies (Ref. 16 and the current work), and three-dimensional cyt b6f structures (2, 3).

Besides modulating electron flow in photosynthesis, the ISP movement has been implicated in redox signaling during state transitions. State transitions are a mechanism for balancing the light absorption capacity of the two photosystems. Moreover, in Chlamydomonas, state transitions are also associated with a switch from linear to cyclic electron flow (for review, see Ref. 17). In chloroplasts, state transitions rely on the reversible phosphorylation of the PSII outer antenna complexes (LHCII) by a kinase that becomes activated when the plastoquinone pool becomes reduced (for review, see Ref. 18). Binding of PQH2 to the Qo site of the b6f complex is required for LHCII kinase activation (19–21). ISP conformational changes have been postulated to play a role in the activation process (21–23). However, the mechanisms that allow transduction of the activating signal from the lumenal to the stromal side of the membrane, where LHCII phosphorylation takes place, are not yet understood. Chlamydomonas LHC kinase has been cloned (24, 25), and a putative transmembrane helix is proposed. This helix might be directly involved in sensing PQH2 binding to the Qo site (22). Nonetheless, the existence of an intrinsic signaling pathway within the b6f complex cannot be excluded.

Here, we performed an extensive mutagenesis study of the chloroplast ISP in Chlamydomonas. Our aim was to compromise its properties by various insertion/deletion/substitution in the flexible hinge, the N-terminal stromal-exposed, and the transmembrane domains and to test the consequences on electron transfer, kinase activation, and cyt b6f complex assembly in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Growth Conditions—Chlamydomonas strains were grown on Tris acetate-phosphate medium at pH 7.2 and 25 °C under 6 microeinsteins m–2 s–1 continuous illumination or in the dark and collected during the exponential phase at 2 x 106 cells ml–1.

Site-directed Mutagenesis and Nuclear Transformation—Plasmid pACR4.5 (ampicillin-sensitive/tetracycline-resistant) containing the Chlamydomonas PetC gene (26) was used as the template for mutagenesis with the oligonucleotides: petC-{Delta}4,

5'-CGTTATGCCGGTGGTGCGCGCgGCCGCTGTGCCCGACATGAACAAGCGCAAC-3'; petC-N17V, 5'-CAAGCGCAACATCATGgtCCTGATCCTGGCTGGCTGGTGGCGCcGGTCTGCCATCAC-3'; petC-A32K, 5'-GCCCATCACCACCCTGaagCTGGGCTACGGcGCCTTCTTCGTGC; petC-+5G, 5'-CTACATTGTTCCTCTTTACAGCTCgGGCggcggtggcggtggcGGTGGCGGCGGTGGCCAG-3'; petC-+3G, 5'-CTACATTGTTCCTCTTTACAGCTCgGGCggcggtggcGGTGGCGGCGGTGGCCAG-3'; petC-+1G, 5'-CATTGTTCCTCTTTACAGCTCgggtGGCGGTGGCGGCGGTGGC-3'; petC-{Delta}1G, 5'-CATTGTTCCTCTTTACAGCTCgGGTGGCGGCGGTGGCCAGGCC-3'; petC-{Delta}3G, 5'-CATTGTTCCTCTTTACAGCTCgGGCGGTGGCCAGGCCGCTAAG-3'.

Mutant plasmids, detected by restoration of ampicillin resistance, were subsequently screened for the NotI (petC-{Delta}4), NarI (petC-N17V, petC-A32K), or AvaI (petC-+5G, petC-+3G, petC-+1G, petC-{Delta}1G, petC-{Delta}3G) restriction sites, underlined in the above oligonucleotide sequences. Chlamydomonas double mutant cells with a cell wall deficiency (cw15) and a deletion in PETC (petC-{Delta}1 in de Vitry et al. (26); here referred to as {Delta}PetC) were transformed as in Kropat et al. (27) using HindIII-linearized plasmids. Phototrophic colonies were selected on minimal medium under light intensities of 40–100 microeinsteins m–2 s–1 and became visible after ~2 weeks. Transformants were characterized as in de Vitry et al. (26) by restriction analysis of specific PCR-amplified products (not shown). Mutations within the Chlamydomonas PetC gene were confirmed by DNA sequencing.

Protein Isolation, Separation, Analysis, and in Vivo Labeling—Thylakoid membranes were purified and resuspended in 10 mM Tricine-NaOH, pH 8.0, containing protease inhibitors (200 µM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 mM {epsilon}-aminocaproic acid) as in Breyton et al. (28). Cyt b6f complexes were extracted by a Hecameg (HG) solubilization (at 30 rather than 25 mM) of thylakoid membranes (29). Polypeptides were separated on 12–18% SDS-polyacrylamide gels containing 8 M urea (30). Lanes were loaded with equal amounts (15 µg) of chlorophyll. Proteins were electrotransferred onto Immobilon NC membranes in a semidry blotting apparatus at 0.8 mA·cm–2 for about 30 min. Immunodetection employed antisera raised against subunits of the Chlamydomonas cyt b6f complex (cyt f, Rieske protein, cyt b6) at a 1/200 dilution, and bands were visualized with 125I-labeled protein A (28). Whole cells grown to 2 x 106 cells/ml were pulse-radiolabeled in the presence of 5 µCi/ml [14C]acetate for either 5 min in the presence of 8 µg/ml cycloheximide (an inhibitor of cytoplasmic translation) or 8 min in the presence of 200 µg/ml chloramphenicol (an inhibitor of chloroplast translation) (30). Labeling was terminated by dilution of the isotope with 10 volumes of chilled 50 mM sodium acetate before thylakoid membrane isolation.

In vivo phosphorylation was performed as described in Wollman and Delepelaire (31) using [33P]orthophosphate instead of [32P]orthophosphate (21). Cells were grown to 2 x 106 cells/ml were harvested and resuspended in a phosphate-depleted medium for 30 min, then 2 µCi/ml [33P]orthophosphate was added for 90 min. Cells were harvested, resuspended in a phosphate-depleted medium, and adapted for 30 min to state I conditions (plastoquinone pool-oxidized; cells were strongly agitated in the dark). To obtain state II conditions (plastoquinone poolreduced), cells were incubated with 5 µM carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP) in the dark without agitation for 30 min (32). Phosphorylation and dephosphorylation reactions were stopped by the addition of 10 mM NaF and rapidly cooled to 4 °C before thylakoid membrane isolation.

Absorption and Fluorescence Spectroscopy—For spectroscopic analysis, cells were collected and re-suspended in 20 mM Hepes-NaOH, pH 7.2, in the presence of 20% Ficoll to prevent cell sedimentation. Measurements were performed at room temperature with a home-built spectrophotometer (33) as described in de Vitry et al. (26). cyt f and b6 reduction was measured as the absorption changes at 554 and 564 nm, respectively, minus a base line drawn between 545 and 573 nm (34). The slow phase (phase b) of the electrochromic shift of carotenoids, which reflects charge transfer across the membrane and cyt b6f catalysis, was measured at 515 nm. Non-saturating actinic flashes were employed to prevent multiple turnovers of the cyt b6f complex. When required, FCCP was added at the concentration of 1 µM to collapse the electrochemical proton gradient.

State I-II transitions were measured according to the procedure previously employed (35); cells were adapted in the dark for 40 min in the presence of glucose oxidase (2 mg/ml), glucose (20 mM), and 3-(3',4'-dichloroprenyl)-1–1-dimethylurea (10 µM), to obtain state II. Cells were then illuminated with continuous light (590 ± 10 nm, 60 microeinsteins m–2 s–1) for 10 min to promote reversal to state I and to probe the state II to I transition. The same cells were then placed into darkness with periodic sampling to probe relaxation back to state II. This procedure allowed a true estimation of the state I-state II transition independent of other metabolic changes that accompany the aerobiosis-anaerobiosis transition.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Location of Mutations in the Rieske Protein N-terminal, Transmembrane, and Hinge Domains—Fig. 1 shows the locations of mutations that were introduced into the Rieske ISP of Chlamydomonas (36) and their alignment with partial sequences of other cyt b6f and bc1 Rieske proteins. Their locations within the N-terminal region of the Chlamydomonas cyt b6f structure (2) are shown in Fig. 2. The first three N-terminal residues (Ala1 to Ala3) and the flexible hinge (Ser43 to Gly50) of the Rieske ISP are not resolved in the structure (2), very likely because of their flexibility, and are not shown in Fig. 2. The eight-residue-long flexible hinge of the Chlamydomonas chloroplast Rieske protein contains two serine residues followed by a stretch of six glycines (Fig. 1). This domain is required for the ISP conformational movement. We modified the length of this domain by the addition of 1, 3, or 5 glycines (mutations +1G, +3G, and +5G) or deletion of 1 or 3 glycines (mutations {Delta}1G and {Delta}3G). In the PetC-{Delta}4 mutant we deleted 4 residues, Ala-Ser-Ser-Glu (ASSE), from the already short N-terminal domain to test the role of this domain in signaling.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 1.
Mutations in the N-terminal, transmembrane, and flexible hinge domains of the Chlamydomonas Rieske ISP and alignment with partial sequences of other ISPs. Shown is deletion of 4 residues in the stromal N terminus ({Delta}4) and diminution of {alpha}-helix polarity (N17V). Charge introduction at lumenal end of the {alpha}-helix (A32K) is indicated by an arrow. Flexible hinge glycine additions (+5G, +3G, +1G) and deletions ({Delta}1G, {Delta}3G) are shown. Transmembrane {alpha}-helices are underlined. The N-terminal {alpha}-helix detected in the Chlamydomonas b6f structure is indicated in gray (2). Flexible hinges are boxed. Ch, C. reinhardtii (36); Sp, spinach; Ma, M. laminosus (3); Sy, Synechococcus sp. PCC 7002; Bo, bovine; Sa, Saccharomyces cerevisiae.

 



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 2.
Mutations in the N-terminal, transmembrane, and flexible hinge of the Rieske protein in the Chlamydomonas b6f complex structure. The N-terminal domain of the Rieske protein (N-ter Rieske) is close to the C-terminal domain of cyt f (C-ter cyt f). An endogenous sulfolipid (SQDG) interacts with the Rieske protein Asn17 and cyt f Lys272 residues.

 
A characteristic feature of the cyt b6f complex is the absence of a charged residue (lysine or arginine) at the lumenal end the Rieske ISP transmembrane helix. Such charged residues at the ends of transmembrane helices serve to anchor these helices to the membrane and are found in the cyt bc1 ISP counterpart (arrow on Fig. 1). To test whether the absence of this charged residue in the cyt b6f ISP represents an important feature for function of the cyt b6f complex, we generated a mutant strain (A32K) where a polar residue has been introduced into the lumenal end of the ISP helix (Figs. 1 and 2). In the cyt b6f structures (2, 3), the N-terminal domain of the Rieske protein lies close to the C-terminal domain of cyt f. In the Chlamydomonas structure, an endogenous sulfolipid (sulfoquinovosyldiacylglycerol (SQDG)) interacts via hydrogen bonds with Rieske protein Arg13 and Asn17 and cyt f Lys272. Residues Arg13 and Asn17 are conserved in cyt b6f Rieske proteins (Fig. 1). To test the role of Asn17 in proton translocation and/or possible assembly-mediated control of cyt f synthesis, we decreased the polarity of this residue by constructing the substitution N17V (see N17V in Figs. 1 and 2).

Rieske ISP Hinge Mutants; Assembly of the cyt b6f Complex and Electron Transfer Properties—To further investigate the relationship between Rieske ISP flexibility and function in the cyt b6f complex, we extended the mutational approach to include insertions of up to 5 residues and deletions of up to 3 residues. Previous ISP hinge mutations (in cyanobacteria) were limited to insertion of 4 residues and deletions of 1 or 2 residues. The two-residue deletion slowed electron transfer by a factor of four (16).

The results of immunodetection analysis indicated that the cyt b6f subunits accumulated to wild type (WT) levels in all of the flexible hinge mutants even though their Rieske proteins were of different size (Fig. 3). Spectroscopic analysis of the modified cyt b6f complex in vivo (Table I) confirmed the high tolerance of the ISP hinge for modifications in length. A dramatic consequence on electron transfer was observed only in the {Delta}3G mutant (three residues deletion of the hinge). The effect of this mutation (Fig. 4; note the differences in times scales on the abscissa between WT in A/B/C and {Delta}3G-1 in D/E/F) was to reduce in a concerted manner the rate of electron injection into both the high potential pathway, as evidenced by the kinetics of cyt f reduction (Fig. 4, panels B and E) and the low potential chain. The latter pathway was monitored by measuring the slow phase of the electrochromic signal (the "b phase," panels A and D), which results from electron transfer between the b hemes. In addition, redox changes of the b hemes were measured directly (panels C and F). Cyt b6 redox kinetics are rather complex, resulting from cyt bL reduction by PQH2 from the Qo-site, cyt bL oxidation by cyt bH, and then cyt bH oxidation by plastoquinone at the Qi site. To directly monitor cyt bL reduction, which is linked to plastoquinol oxidation, kinetics were measured in the presence of the inhibitor 2-N-4-hydroxyquinoline-N-oxide. This compound selectively slows down cyt bH oxidation at the Qi site (37) and allows a correct deconvolution of the reduction signal (triangles in panels C and F).



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 3.
Accumulation of cyt b6f subunits in the Rieske ISP flexible hinge mutants. Cells from the exponential phase of growth were analyzed by SDS-PAGE (12–18% acrylamide, 8 M urea). Cyt f and Rieske protein were immunodetected. WT and Rieske deletion mutant ({Delta}petC) strains are shown as controls.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Functional properties of the Rieske ISP mutants

ND, not determined.

 



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 4.
Kinetics of the electrochromic signal (A and D), cyt f (B and E), and cyt b6 (C and F) redox changes in the WT (A, B, and C) and {Delta}3G mutant (D, E, and F). Algae were collected during exponential growth phase and resuspended in Hepes 20 mM, pH 7.2, buffer with the addition of 20% (w/v) Ficoll to prevent sedimentation. Cells were illuminated with non-saturating actinic flashes of red light at a frequency of 0.15 Hz. 3-(3',4'-dichloroprenyl)-1–1-dimethylurea and hydroxylamine were added at concentrations of 10 µM and 1 mM, respectively, to block PSII activity. Where indicated, FCCP was added at the concentration of 1 µM to facilitate pH equilibration between the chloroplast compartments. 2-N-4-hydroxyquinoline-N-oxide (NQNO) was added at the concentration of 4 µM to slow plastoquinone reduction at the Qi site. The fast and slow phases of the electrochromic signal in panels A and D represent charge separation within PSI and the cyt b6f complex, respectively. In panels B, C, E, and F, upward and downward absorption changes correspond to reduction and oxidation of the cyt b6f redox cofactors, respectively. –{Delta}I/I is the difference between the light transmitted through the measuring and reference cuvettes divided by the light transmitted through the reference cuvette.

 
It is known that a proton gradient exists across the thylakoid membranes of dark adapted Chlamydomonas cells. This gradient arises from ATP hydrolysis by the CF0-F1 ATP synthase-ATPase complex (34). The gradient slows down the kinetics of plastoquinol oxidation at the Qo site. In the {Delta}3G mutant, the consequences of the electrochemical proton gradient on cyt b6f catalysis were larger than in the wild type; the rates of PQH2 oxidation were 12 and 50 times slower than in wild type cells in the presence and absence of the protonophore FCCP, respectively. This phenotype resembles that of two other cyt b6f mutants of Chlamydomonas; FUD2, in which the Qo site of the cyt b6f is modified because of a 36-base pair duplication in the cd loop of cyt b6 (34), and ycf7, where the PetL subunit is disrupted (38). Both of these mutants show loosened binding of the Rieske protein to the cyt b6f complex. We, therefore, tested whether a similar perturbation of ISP binding could be observed in the {Delta}3G mutant. To this aim, cyt b6f complexes were purified from exponentially growing cells of the {Delta}3G mutant using the Hecameg solubilization procedure described in Pierre et al. (29). The distributions of cyt f and Rieske proteins upon gradient centrifugation peaked in the same fractions and were similar for the {Delta}3G and wild type strains (Fig. 5). These results indicate that Rieske protein binding to the b6f complex was not loosened in the {Delta}3G mutant. Therefore, the large sensitivity of the electron transfer rate to an electrochemical proton gradient appears to have a different physical basis in the {Delta}3G mutant than in the FUD2/ycf7mutants.



View larger version (57K):
[in this window]
[in a new window]
  		FIG. 5.
Binding of the Rieske protein to the b6f complex is not loosened in the {Delta}3G mutant. Thylakoid membranes were solubilized with HG. The supernatant was centrifuged on a 10–30% sucrose gradient. Fractions of the gradient were collected, analyzed by SDS-PAGE (12–18% acrylamide, 8 M urea), and immunodetected.

 
Rieske ISP Hinge Mutants; Structural Changes and LHC Kinase Activation—The strong kinetic effect measured in the {Delta}3G mutant, which has a catalytic rate 50 times slower than the wild type in the presence of the electrochemical proton gradient, offered an opportunity to test the possible consequences of a decreased hinge flexibility on the efficiency of LHC kinase activation. To perform such a study, fluorescence emission was measured at room temperature in intact algae upon adaptation to anaerobiosis. 3-(3',4'-Dichloroprenyl)-1–1-dimethylurea was added to prevent PSII photochemistry. This allowed an estimation of the fraction of antenna connected to PSII directly from fluorescence emission changes. Algae in state II (see "Materials and Methods") were submitted to illumination to promote transition to state I. Subsequently, their recovery to state II was followed in the dark. This procedure was adopted (35) to distinguish between the intrinsic state I-state II transition rate and that of plastoquinone reduction, which often limits the overall process of state II transition upon adaptation of the algae to anaerobiosis.

No significant differences were observed in the ability to perform a state I-state II transition of the wild type and the {Delta}3G mutant (we calculated values of 60 ± 22 s for this transition in the wild type (mean of 6 different experiments) and 72 ± 14 s (4 experiments) for the {Delta}3G mutant) nor of any of the other ISP hinge mutants (not shown). No state II transition was observed in the {Delta}petC strain (lacking the chloroplast ISP), likely because of its blockage in plastoquinol binding. Consistent with the fluorescence measurements, a wild type pattern of antenna protein phosphorylation was observed in the {Delta}3G and +5G mutants (Fig. 6). In contrast and regardless of the state condition, the {Delta}petC mutant displayed a low and constant level of phosphorylation of LHC proteins CP26, CP29, and p11 and an absence of phosphorylation of LHC proteins p13, p17, and cyt b6f subunit PetO (39)). This condition is typical of state I.



View larger version (67K):
[in this window]
[in a new window]
  		FIG. 6.
Autoradiogram of 33P-radiolabeled polypeptides. Protein are shown in the 15–35-kDa range. A, thylakoid membrane proteins of WT strain and Rieske protein deletion mutant ({Delta}petC). B, thylakoid membrane proteins of Rieske protein hinge insertion (+5G) and deletion ({Delta}3G) mutants. Cells were placed in state I (I) or state II (II) conditions. Stars indicate the absence of phosphorylation.

 
Rieske Protein Mutants in the Stromal and Transmembrane Domains—Neither the {Delta}4 mutant (deletion of 4 residues from the N-terminal domain) nor the A32K mutant (insertion of a polar residue at the lumenal end of the ISP transmembrane helix) displayed any remarkable phenotype with respect to accumulation of the cyt b6f complex (Fig. 7), electron transport (Table I), or state transitions (not shown). On the other hand a significant decrease in the accumulation of cyt b6f subunits was observed in both the {Delta}petC and the N17V mutants (Fig. 7A). The rates of synthesis and membrane insertion during a 5-min pulse labeling of cyt b6 and subunit IV were very similar in the N17V and {Delta}PetC mutants and the wild type (Fig. 7C). The rate of synthesis and membrane insertion of the Rieske ISP during an 8-min pulse-labeling showed no differences between the wild type and the N17V mutant but showed an absence of label incorporation into Rieske protein (as expected) in the {Delta}petC mutant (Fig. 7B). Still, the lower accumulation of the Rieske protein in the N17V mutant was not due to a higher proteolytic susceptibility of the assembled Rieske protein since it remained as stable in N17V as in wild type over 6 h, in an immunodetection experiment where the amount of Rieske protein at various time points after the addition of an inhibitor of cytoplasmic translation was probed in vivo with specific antibodies (not shown). These observations argue for a rapid degradation of a fraction of neosynthesized Rieske before its assembly in cyt b6f complexes in the N17V mutant.



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 7.
Accumulation and synthesis rates of cyt b6f subunits in Rieske ISP N-terminal and transmembrane domain mutants. A, immunodetection of cyt f and Rieske protein in mutant cells from exponential growing phase; accumulation of the {alpha} subunit of CF1 provided a loading control. B, cytoplasmic pulse-labeled, translation products. C, chloroplast pulse-labeled translation products. Whole cells from Rieske ISP deletion ({Delta}petC) and N17V substitution (N17V) mutants, WT, and cyt b6-deficient ({Delta}petB) mutant strains were pulse-labeled with [14C]acetate for either 8 min in the presence of an inhibitor of chloroplast translation or for 5 min in the presence of an inhibitor of cytoplasmic translation. Thylakoid membrane proteins were separated by SDS-PAGE (12–18% acrylamide, 8 M urea). Stars indicate the absence or large decrease of rate of synthesis of a translation product. Black squares indicate translation products of the major b6f subunits. Psb defines various PSII subunits.

 
In {Delta}petB (lacking cyt b6), {Delta}petC, and N17V mutants, there was a large decrease in the rate of cyt f synthesis, down to the limit of detection (see the stars in Fig. 7C). The rate of cyt f synthesis decreased by a factor of 3 in the {Delta}petC and N17V mutants as compared with a factor of 10 in the {Delta}petB mutant. This has been attributed previously to an assembly-mediated control of cyt f synthesis (40). These data reveal a new role for the Rieske ISP transmembrane helical domain; namely, as an additional partner involved in assembly-mediated control of cyt f synthesis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The Rieske Fe2S2 protein is actively involved in forming the quinol binding site in both bc1 and b6f cyt complexes. Moreover, it plays a direct role in catalysis by means of the large movement of the Rieske cluster domain, which is linked to the membrane via a flexible hinge. The same movement has been implicated in the transduction of the activating signal from the Qo site to the LHCII kinase during state transitions. In the current work we have addressed in detail the involvement of the Rieske protein in these two major processes by generating site-specific mutations in the flexible hinge, the transmembrane helix, and the N-terminal, stromal-exposed domain. We have also investigated the role of the Rieske ISP in regulation of the assembly of the cyt b6f complex.

The Flexible Hinge of the b6f Rieske Protein Is More Tolerant to Insertions than Deletions—The b6f Rieske ISP hinge of Chlamydomonas contains a string of six glycines. We have varied the number of consecutive glycines in the hinge from 11 (+5G) to 3 ({Delta}3G). All the generated mutants correctly assembled the cyt b6f complex, and only one of them ({Delta}3G) showed a reduction in the catalytic efficiency of the complex. This supports and extends previous findings, indicating a large tolerance of the flexible hinge of the b6f Rieske protein to structural changes (16, 26). At the same time we show here that it is indeed possible to severely lower the efficiency of electron flow in the cyt b6f complex by a modification of the Rieske hinge. The {Delta}3G mutation slowed catalysis by a factor of ~10-fold. Consistent with the severe consequences of extended deletions, we could not select by restoration of phototrophy a {Delta}5G mutation (data not shown). Therefore, despite the greater flexibility of the cyt b6f Rieske protein hinge, modifications that prevent ISP conformational changes have clearly deleterious consequences on catalytic efficiency in both b6f and bc1 cyt complexes. These data are consistent with a requirement for Rieske ISP conformational mobility in cyt b6f complexes.

The b6f ISP subunit can tolerate large modifications of its flexible hinge, particularly increases in length, without compromising catalytic efficiency (Refs. 16 and 26 and this work). This is not the case in the bc1 ISP counterpart (for review, see Ref. 11). The differing degrees of flexibility of the Rieske ISP hinge observed in bc1 and b6f complexes are consistent with the three-dimensional structures of these complexes. In the former the hinge is clearly defined. It is extended when the Fe2S2 cluster resides in the proximal (Qo) position and in a helical conformation when the cluster occupies the distal (cyt c1) position (7–9, 41). No structures are available for different conformations of the cyt b6f Rieske ISP subunit. Nevertheless, the hinge region is clearly less well defined. The hinge assumes an intermediate conformation between the proximal and the distal positions in one of the available structures (3) and is not resolved in the other (2) because of a very large degree of freedom.

The Rieske Protein Is Required for Kinase Activation, but Its Rapid Movement Is Not Limiting—The Rieske ISP deletion mutant {Delta}petC is locked in a state I condition. This likely arises from a nonfunctional Qo site that, therefore, does not allow activation of the LHCII kinase. This phenotype is in agreement with previous findings, showing that binding of plastoquinol to the Qo site is essential for signal transduction during state transitions (19–21).

In contrast, the {Delta}3G mutant, where plastoquinol oxidation was very slow because of the truncated ISP hinge, did not show any significant alteration in the kinetics of state I-state II transitions. In principle, this result might suggest that the movement of the Rieske ISP does not play any role in state transitions. However, even in the {Delta}3G mutant, the rate of electron transfer (and, therefore, the rate of Rieske ISP conformational changes) is at least 2 orders of magnitude faster than the rate of the state I-state II transition itself. This rate may be governed primarily by the kinetic properties of the kinase itself (35), although LHCII lateral diffusion in thylakoid membranes has also been implicated (see e.g. Ref. 42). Therefore, we conclude that as long as conformational changes related to plastoquinol binding to the Qo site remain faster than the limiting step of state transitions, the latter process can take place efficiently even in the presence of a severely crippled Qo site. This is consistent with the phenotype of the Chlamydomonas FUD2 mutant, where electron flow is slowed ~8-fold by a reduced binding affinity of plastoquinol for the Qo site, but state transitions remain unaffected (34).

A central, unresolved issue in chloroplast state transitions and perhaps redox signaling more generally is the pathway employed to transduce the activating signal from the lumen, where plastoquinol binds to the Qo site, to the stroma, where the catalytic domain of the kinase resides. Several hypotheses have been formulated. (i) An LHCII kinase might be activated through a direct interaction between its transmembrane helix and the Qo site (22). (ii) A transmembrane conformational change within the b6f complex might carry the signal (23), as supported by subunit IV-PetL fusion experiments (43), (iii) perhaps via chlorophyll or {beta}-carotene molecules, as suggested in Stroebel et al. (2), or (iv) alternatively, via conformational changes within the Rieske ISP itself. In this study, we have rendered the latter hypothesis less likely by modifying several domains of the Rieske ISP without altering state transitions.

Role of the Rieske ISP and a Sulfolipid in Assembly-mediated Control of cyt f Synthesis—We have previously shown that the Rieske protein deletion mutant {Delta}petC accumulates a lower amount of cyt b6f complex than the wild type (26). The steady state accumulation of cyt b6f complex in the {Delta}petC mutant is increased when the ClpP protease expression is attenuated (44). Therefore, part of the diminished level of cyt b6f accumulation can be attributed to proteolytic degradation. In addition, cyt f accumulation is regulated by an "assembly-mediated control" process that results in markedly decreased cyt f synthesis in the absence of the large, chloroplast-encoded cyt b6 and subunit IV components of the cyt b6f complex (40). This process, termed CES (control by epistasy of synthesis), occurs by the autoregulation of cyt f translation via the 5'-untranslated region of petA (cyt f) mRNA (45) and the cyt f protein C-terminal domain (46). The proximity of the N-terminal Rieske ISP transmembrane helix and the cyt f C-terminal transmembrane helix in the cyt b6f structures (2, 3) suggested that the Rieske protein might also play a more direct role in this process. Consistent with this idea, we observed a 3-fold decrease of cyt f synthesis in the Rieske-protein–mutant, {Delta}petC, similar to that reported previously (47). This decrease is less pronounced than the 10-fold decrease in cyt f synthesis observed in the absence of the cyt b6 and subunit IV proteins (40) but clearly points to a role for the Rieske protein in assembly-mediated control of cyt f synthesis.

The rate of synthesis of the Rieske protein is not affected in N17V mutant. The lower accumulation of the N17V Rieske protein should be attributed to a rapid degradation of unassembled ISP rather than to a higher susceptibility to proteases of the assembled N17V variant of the Rieske ISP that proved as stable as in the wild type over 6 h. The lower rate of cyt f synthesis in the N17V mutant suggests that, although most of the neosynthesized Rieske ISP assembles in cyt b6f complexes, there is a modified conformation of its transmembrane helix due to the N17V substitution that allows assembled cyt f to exert some negative control of its own synthesis. Indeed, the Chlamydomonas b6f structure reveals the presence of an endogenous sulfolipid SQDG that interacts with Rieske protein residues Arg13 and Asn17 and cyt f Lys272 (see Fig. 2). Lys272 has been established as a key residue in the control of cyt f synthesis (46). The R13K mutation (48) is semiconservative; it substitutes a basic residue by another basic residue that should still allow the hydrogen bond formation with SQDG, and it did not affect the rate of cyt f synthesis (not shown). In contrast, N17V mutation substitutes a polar residue by a non-polar residue, which prevents the hydrogen bond formation with SQDG; in N17V mutant, the rate of cyt f synthesis is clearly decreased. This suggests that the region delimited by the endogenous sulfolipid, the Rieske protein, and the cyt f helix plays a specific role in the assembly-mediated control of cyt f synthesis. It is tempting to speculate that removal of the Rieske protein or modification of its binding to the sulfolipid would increase the accessibility of the cyt f target domain to the trans-acting factors that regulate translation of cyt f (46, 49).

Sulfolipids are known to play major roles in photosynthetic membranes. The sulfolipid SQDG is crucial for photosynthesis in Synechocystis sp. PCC 6803 (50). The growth of a sulfolipid-deficient Arabidopsis mutant is impaired after phosphate starvation (51). A double mutant of Arabidopsis lacking SQDG and phosphatidylglycerol is impaired in PSII activity and photosynthetic growth (52). Several Chlamydomonas mutants lacking SQDG have altered PSII activity or structural integrity (53–55) including the D1 protein as a major target (56). Therefore, SQDG seems to be involved in the turnover of both D1 and cyt f. The synthesis of both subunits is modulated by the presence of their assembly partners. This raises the interesting question of whether a similar mechanism underlies the role of SQDG in the assembly of both subunits and, as a consequence, in the operation of the CES process.


   FOOTNOTES
 
* This work was supported by the CNRS, National Science Foundation Grant MCB 0091415, and University of Wisconsin-Oshkosh Faculty Development R788 grants (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 33-1-58-41-50-55; Fax: 33-1-58-41-50-22; E-mail: catherine.devitry{at}ibpc.fr.

1 The abbreviations used are: cyt, cytochrome; HG, Hecameg (6-O-(N-heptylcarbamoyl)-methyl-{alpha}-D-glycopyranoside); ISP, iron-sulfur protein; LHC, light-harvesting complex; PQH2, plastoquinol; PSI, photosystem I; PSII, photosystem II; SQDG, sulfoquinovosyldiacylglycerol; WT, wild type; FCCP, carbonylcyanide-p-(trifluoromethoxy)phenylhydrazine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge C. Facciotti as a recipient of a European Economic Community Erasmus scholarship for participation in Rieske ISP mutagenesis, F. Zito for in vivo phosphorylation labeling expertise, D. Picot and D. Stroebel for stimulating discussions and privileged communication of the Chlamydomonas cyt b6f structure, and Y. Choquet for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Berry, E. A., Guergova-Kuras, M., Huang, L.-S., and Crofts, A. R. (2000) Annu. Rev. Biochem. 69, 1005–1075[CrossRef][Medline] [Order article via Infotrieve]
  2. Stroebel, D., Choquet, Y., Popot, J.-L., and Picot, D. (2003) Nature 426, 413–418[CrossRef][Medline] [Order article via Infotrieve]
  3. Kurisu, G., Zhang, H., Smith, J. L., and Cramer, W.A. (2003) Science 302, 1009–1014[Abstract/Free Full Text]
  4. de Vitry, C., Desbois, A., Redeker, V., Zito, F., and Wollman, F.-A. (2004) Biochemistry 43, 3956–3968[CrossRef][Medline] [Order article via Infotrieve]
  5. Mitchell, P. (1975) FEBS Lett. 59, 137–139[CrossRef][Medline] [Order article via Infotrieve]
  6. Crofts, A. R., Meinhardt, S. W., Jones, K. R., and Snozzi, M. (1983) Biochim. Biophys. Acta 723, 202–218
  7. Xia, D., Yu, C.-A., Kim, H., Xia, J.-Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1997) Science 277, 60–66[Abstract/Free Full Text]
  8. Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y.-I., Kim, K. K., Hung, L.-W., Crofts, A. R., Berry, E. A., and Kim, S.-H. (1998) Nature 392, 677–684[CrossRef][Medline] [Order article via Infotrieve]
  9. Iwata, S., Lee, J. W., Okada, K., Lee, J. K., Iwata, M., Rasmussen, B., Link, T., Ramaswamy, S., and Jap, B. K. (1998) Science 281, 64–71[Abstract/Free Full Text]
  10. Brugna, M., Rodgers, S., Schricker, A., Montoya, G., Katzmeier, M., Nitschke, W., and Sinning, I. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2069–2074[Abstract/Free Full Text]
  11. Darrouzet, E., Moser, C.C., Dutton, P.L., and Daldal, F. (2001) Trends Biochem. Sci. 26, 445–451[CrossRef][Medline] [Order article via Infotrieve]
  12. Breyton, C. (2000) Biochim. Biophys. Acta 1459, 467–474[Medline] [Order article via Infotrieve]
  13. Heimann, S., Ponamarev, M. V., and Cramer, W. A. (2000) Biochemistry 39, 2692–2699[CrossRef][Medline] [Order article via Infotrieve]
  14. Soriano, G. M. Guo, L. W., de Vitry, C., Kallas, T., and Cramer, W. A. (2002) J. Biol. Chem. 277, 1865–1871
  15. Schoepp, B., Brugna, M., Riedel, A., Nitschke, W., and Kramer, D. M. (1999) FEBS Lett. 450, 245–250[CrossRef][Medline] [Order article via Infotrieve]
  16. Yan, J., and Cramer, W. A. (2003) J. Biol. Chem. 278, 20925–20933[Abstract/Free Full Text]
  17. Wollman, F. A. (2001) EMBO J. 20, 3623–3630[CrossRef][Medline] [Order article via Infotrieve]
  18. Allen, J. F. (1992) Biochim. Biophys. Acta 1098, 275–335[Medline] [Order article via Infotrieve]
  19. Vener, A. V., van Kan, P. J., Gal, A., Andersson, B., and Ohad, I. (1995) J. Biol. Chem. 270, 25225–25232[Abstract/Free Full Text]
  20. Vener, A. V., van Kan, P. J., Rich, P., Ohad, I., and Andersson, B. (1997) Proc. Natl. Sci. Acad. U. S. A. 94, 1585–1590[Abstract/Free Full Text]
  21. Zito, F., Finazzi, G., Delosme, R., Nitschke, W., Picot, D., and Wollman, F.-A. (1999) EMBO J. 18, 2961–2969[CrossRef][Medline] [Order article via Infotrieve]
  22. Vener, A. V., Ohad, I., and Andersson, B. (1998) Curr. Opin. Plant Biol. 1, 217–223[CrossRef][Medline] [Order article via Infotrieve]
  23. Finazzi, G., Zito, F., Barbagallo, P. R., and Wollman, F.-A. (2001) J. Biol. Chem. 276, 9770–9774[Abstract/Free Full Text]
  24. Fleischmann, M. M., Ravanel, S., Delosme, R., Olive., J., Zito, F, Wollman, F. A., and Rochaix J.-D. (1999) J. Biol. Chem. 274, 30987–30994[Abstract/Free Full Text]
  25. Depège, N., Bellafiore, S., and Rochaix, J.-D. (2003) Science 299, 1572–1575[Abstract/Free Full Text]
  26. de Vitry, C., Finazzi, G. Baymann, F., and Kallas, T. (1999) Plant Cell 11, 2031–2044[Abstract/Free Full Text]
  27. Kropat, J., von Gromoff, E. D., Müller, F. W., and Beck, C. F. (1995) Mol. Gen. Genet. 248, 727–734[CrossRef][Medline] [Order article via Infotrieve]
  28. Breyton, C., de Vitry, C., and Popot, J.-L. (1994) J. Biol. Chem. 269, 7597–7602[Abstract/Free Full Text]
  29. Pierre, Y., Breyton, C., Kramer, D., and Popot, J.-L. (1995) J. Biol. Chem. 270, 29342–29349[Abstract/Free Full Text]
  30. Lemaire, C., Girard-Bascou, J., Wollman, F.-A., and Bennoun, P. (1986) Biochim. Biophys. Acta 851, 229–238
  31. Wollman, F.-A., and Delepelaire, P. (1984) J. Cell Biol. 98, 1–7[Abstract/Free Full Text]
  32. Bulté, L., Gans, P., Rebéillé, F., and Wollman, F.-A. (1990) Biochim. Biophys. Acta 1020, 72–80
  33. Joliot, P., and Joliot, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1034–1038[Abstract/Free Full Text]
  34. Finazzi, G., Büschlen, S., de Vitry, C., Rappaport, F., Joliot, P., and Wollman, F.-A. (1997) Biochemistry 36, 2867–2874[CrossRef][Medline] [Order article via Infotrieve]
  35. Delepelaire, P., and Wollman, F.-A. (1985) Biochim. Biophys. Acta 809, 277–283
  36. de Vitry, C. (1994) J. Biol. Chem. 269, 7603–7609[Abstract/Free Full Text]
  37. Selak, M. A., and Whitmarsh, J. (1982) FEBS Lett. 150, 286–292
  38. Takahashi, Y., Rahire, M., Breyton, C., Popot, J.-L., Joliot, P., and Rochaix, J.-D. (1996) EMBO J. 15, 3498–3506[Medline] [Order article via Infotrieve]
  39. Hamel, P., Olive, J., Pierre, Y., Wollman, F.-A., and de Vitry, C. (2000) J. Biol. Chem. 275, 17072–17079[Abstract/Free Full Text]
  40. Kuras, R., and Wollman, F.-A. (1994) EMBO J. 13, 1019–1027[Medline] [Order article via Infotrieve]
  41. Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H. (2000) Structure 8, 669–684[Medline] [Order article via Infotrieve]
  42. Drepper, F., Carlberg, I., Andersson, B., and Haehnel, W. (1993) Biochemistry 32, 11915–11922[CrossRef][Medline] [Order article via Infotrieve]
  43. Zito, F., Vinh, J., Popot, J.-L., and Finazzi, G. (2002) J. Biol. Chem. 277, 12446–12455[Abstract/Free Full Text]
  44. Majeran, W., Wollman, F.-A., and Vallon, O. (2000) Plant Cell 12, 137–149[Abstract/Free Full Text]
  45. Choquet, Y., Stern, D. B., Wostrikoff, K., Kuras, R., Girard-Bascou, J., and Wollman, F.-A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4380–4385[Abstract/Free Full Text]
  46. Choquet, Y., Zito, F., Wostrikoff, K., and Wollman, F.-A. (2003) Plant Cell 15, 1445–1454
  47. Majeran, W. (2002) La Protéolyse dans le Chloroplaste de Chlamydomonas reinhardtii: Rôle et Organization Structurale de la Protéase ClpP. Ph.D thesis, Institut National Agronomique Paris-Grignon
  48. Finazzi, G., Chasen, C., Wollman, F.-A., and de Vitry, C. (2003) EMBO J. 22, 807–815[CrossRef][Medline] [Order article via Infotrieve]
  49. Wostrikoff, K., Choquet, Y., Wollman, F.-A., and Girard-bascou, J. (2001) Genetics 159, 119–132[Abstract/Free Full Text]
  50. Aoki, M., Sato, N., Meguro, A., and Tsuzuki, M. (2004) Eur. J. Biochem. 271, 685–693[Medline] [Order article via Infotrieve]
  51. Yu, B., Xu, C., and Benning, C. (2002) Proc. Natl. Sci. Acad. U. S. A. 99, 5732–5737[Abstract/Free Full Text]
  52. Yu, B., and Benning, C. (2003) Plant J. 36, 762–770[CrossRef][Medline] [Order article via Infotrieve]
  53. Sato, N., Sonoike, K., Tsuzuki, M., and Kamaguchi, A. (1995) Eur. J. Biochem. 234, 16–23[Medline] [Order article via Infotrieve]
  54. Riekhof, W. R., Ruckle, M. E., Lydic, T. A., Sears, B. B., and Benning, C. (2003) Plant Physiol. 133, 864–874[Abstract/Free Full Text]
  55. Sato, N., Aoki, M., Maru, Y., Sonoike, K., Minoda, A., and Tsuzuki, M. (2003) Planta 217, 245–251[Medline] [Order article via Infotrieve]
  56. Minoda, A., Sonoike, K., Okada, K., Sato, N., and Tsuzuki, M. (2003) FEBS Lett. 553, 109–112[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/43/44621    most recent
M406955200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by de Vitry, C.
Right arrow Articles by Kallas, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Vitry, C.
Right arrow Articles by Kallas, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement