Arabidopsis Tic62 and ferredoxin-NADP(H) oxidoreductase form light-regulated complexes that are integrated into the chloroplast redox poise

Abstract

Translocation of nuclear-encoded preproteins across the inner envelope of chloroplasts is catalyzed by the Tic translocon, consisting of Tic110, Tic40, Tic62, Tic55, Tic32, Tic20, and Tic22. Tic62 was proposed to act as a redox sensor of the complex because of its redox-dependent shuttling between envelope and stroma and its specific interaction with the photosynthetic protein ferredoxin-NADP(H) oxidoreductase (FNR). However, the nature of this close relationship so far remained enigmatic. A putative additional localization of Tic62 at the thylakoids mandated further studies examining how this feature might be involved in the respective redox sensing pathway and the interaction with its partner protein. Therefore, both the association with FNR and the physiological role of the third, thylakoid-bound pool of Tic62 were investigated in detail. Coexpression analysis indicates that Tic62 has similar expression patterns as genes involved in photosynthetic functions and protein turnover. At the thylakoids, Tic62 and FNR form high molecular weight complexes that are not involved in photosynthetic electron transfer but are dynamically regulated by light signals and the stromal pH. Structural analyses reveal that Tic62 binds to FNR in a novel binding mode for flavoproteins, with a major contribution from hydrophobic interactions. Moreover, in absence of Tic62, membrane binding and stability of FNR are drastically reduced. We conclude that Tic62 represents a major FNR interaction partner not only at the envelope and in the stroma, but also at the thylakoids of Arabidopsis thaliana and perhaps all flowering plants. Association with Tic62 stabilizes FNR and is involved in its dynamic and light-dependent membrane tethering.

Figure 1.

Localization of Tic62 and FNR in Arabidopsis Chloroplasts. Tic62 and FNR show a triple localization in chloroplasts. Immunoblot analysis of Arabidopsis chloroplast subfractions envelope (Env), stroma (Str), and thylakoids (Thy) using antibodies generated against Tic62, FNR, FBPase (stroma marker), OE23 (thylakoid marker; soluble lumenal protein and thus also detected in minor amounts in the stroma), and OEP16 (envelope marker).

Figure 2.

Thylakoidal Tic62 Comigrates Almost Exclusively with FNR. (A) Comigration of Tic62 and FNR was observed in 2D-BN/SDS-PAGE of pea thylakoids solubilized with 1% n-dodecyl β-d-maltoside. The first dimension (10 μg chlorophyll) and immunoblots of the second dimension with Tic62 and FNR antibodies are shown with the positions of the major thylakoidal complexes indicated. Red lines represent the main signals detected for Tic62 and the dashed line the location of PSI, which displays a slower mobility than Tic62 and FNR. (B) Tic62 does not comigrate with the high molecular weight NDH complex or the Cytb₆f complex. The first dimension and immunoblots of the second dimension of 2D-BN/SDS-PAGE of Arabidopsis chloroplasts (20 μg chlorophyll) with Tic62 and Ndh-H antibodies are shown (top panel). The NDH complex was detected in two complexes, probably representing a dimeric and a monomeric form. The immunoblot of the second dimension of a 2D-BN/SDS-PAGE of pea thylakoids is depicted (bottom panel), incubated with antibodies generated against Tic62 and Cytf (PetA). The Cytb₆f complex is found in a monomeric (Cytb₆f-M) and a dimeric (Cytb₆f-D) form. Positions of molecular weight marker bands in the first and second dimensions are indicated (in kD) as well as the acrylamide concentration gradient used for the BN-PAGEs.

Figure 3.

Tic62 Knockout Lines Display a Specific Reduction of FNR in the Membrane Fractions. (A) Genomic structure of Tic62 from Arabidopsis (At3g18890). Black boxes denote exons, black lines introns, and dotted lines 5′ and 3′ untranslated regions (not to scale). The insertion sites of T-DNAs in lines SAIL_124G04 (tic62-1) and GABI_439H04 (tic62-2) are indicated by triangles. Furthermore, binding sites for Tic62 gene-specific primers and T-DNA–specific left border primers used for screening for homozygous plants are depicted. (B) Tic62 transcript is almost completely absent in tic62 knockout lines. An RT-PCR experiment performed with the Ex3fwd-Ex7rev primers (being 3′ of the tic62-2 and flanking the tic62-1 T-DNA insertion site) of the wild type, tic62-1, and tic62-2 RNA is shown. (C) No Tic62 protein can be detected in tic62 mutants by an immunoblot analysis of wild-type, tic62-1, and tic62-2 chloroplast extracts. Note that the Arabidopsis Tic62 protein displays an aberrant mobility and is found at ∼98 kD in SDS-PAGE gels. (D) FNR is specifically lost from membrane fractions of tic62 chloroplasts. Five micrograms of protein were used for envelope and thylakoid fractions and 10 μg protein in stroma samples. The immunoblot shows the signals obtained with antibodies generated against Tic62, FNR, FBPase (stroma marker), OE23 (thylakoid marker, soluble lumenal protein), and OEP16 (envelope marker) of envelope, stroma, and thylakoids from wild-type and tic62 chloroplasts. Beneath the blot the quantification of FNR in the various fractions is shown, in which the wild-type amount was arbitrarily set to 100% (±sd; n = 2). (E) Tic subunits are not affected in tic62 plants. Immunoblot of wild-type and tic62 envelopes with Tic110, Tic55, Tic40, and Tic32 antibodies. (F) Tic62 is located at the stroma-lamellae of thylakoids. Arabidopsis thylakoids from wild-type and tic62 plants were subfractionated by differential centrifugation. A Coomassie-stained 15% urea-SDS gel (major thylakoid proteins are indicated) and an immunoblot of the obtained fractions is shown: T, untreated thylakoids; 10K, centrifugation at 10,000g, containing grana thylakoids; 40K, centrifugation at 40,000g, representing margins; 140K, centrifugation at 140,000g, enriching stroma lamellae. SN represents the final trichloroacetic acid-precipitated supernatant. For the immunoblots, 5 μg of chlorophyll and 10 μg protein of the supernatant were used per lane and probed with antibodies against Tic62, FNR, and representatives of the main thylakoidal protein complexes: PSII (D1), PSI (PsaF), NDH (Ndh-H), and the ATPase (CF1αβ). The indicated chlorophyll a:b ratio is a measure for the successful enrichment of grana (low a:b ratio) or stroma (high a:b ratio) thylakoids. The figure shows one of two independent repetitions with essentially identical results.

Figure 4.

The Loss of Tic62 Does Not Affect FNR Import but Inhibits the Formation of FNR HMW Complexes. (A) Import of pLFNR1 is not affected in tic62 chloroplasts. Import into isolated wild-type and tic62 Arabidopsis chloroplasts was started by the addition of translation product (pLFNR1 as well as pLHCB1.3 and pGAP-B as controls) and performed for the indicated time. Import products, including 10% of translation product (TL), were separated by SDS-PAGE, radiolabeled proteins analyzed by a phosphor imager, and the signals of the processed mature forms quantified (±sd; n = 3). p, precursor form; m, mature form; a.u., arbitrary units. (B) LFNR1 integration into HMW complexes is defective in tic62 thylakoids. The pLFNR1 protein (and pLHCB1.3 as control) was first imported into wild-type and tic62 chloroplasts for 30 min, and the membranes were subsequently separated from the stroma compartment by disruption of the chloroplasts and centrifugation. The resulting fractions of supernatant (S) and pellet (P) were separated by a BN-PAGE gel (5 to 12% acrylamide) and analyzed on a phosphor imager. The approximate position of BN size markers is indicated, and the HMW Tic62/FNR complexes are marked by an asterisk. (C) The amount of Tic62 is reduced in lfnr1 and lfnr2 plants. An immunoblot with αTic62 and αFNR antibodies of total protein extract from wild-type, tic62, lfnr1, and lfnr2 plants is shown. Ten micrograms of total leaf protein extract was used in SDS-PAGE and 30 μg of protein for native PAGE. (D) The HMW Tic62/FNR complexes are similarly absent in tic62, lfnr1, and lfnr2 thylakoids. BN-PAGE (5 to 13.5%) of thylakoids isolated from wild-type, tic62, lfnr1, and lfnr2 plants. An unstained gel lane indicating the major thylakoidal complexes and immunoblots with αTic62 and αFNR antibodies is shown. The approximate position of BN size markers is indicated, and the HMW Tic62/FNR complexes are marked by an asterisk. [See online article for color version of this figure.]

Figure 5.

Membrane Attachment of the FNR/Tic62 Complexes Is Regulated by Light and Stromal pH. (A) The amount of thylakoid-bound Tic62/FNR complexes is dependent on ambient growth light of the plants. Thylakoids were prepared from dark-adapted plants and compared with growth light (GL; 4 h under 100 μmol photons m⁻² s⁻¹) or high light (HL; 2 h under 1000 μmol photons m⁻² s⁻¹) treated samples. A BN-PAGE gel lane (5 to 13.5%) from wild-type and tic62 thylakoids with the major photosynthetic protein complexes indicated and immunoblots with αTic62 and αFNR antibodies are shown. The approximate position of BN size markers is indicated, and the HMW Tic62/FNR complexes are marked by an asterisk. (B) The attachment of Tic62 and FNR to thylakoids is dependent on the stromal pH. Isolated pea thylakoids were mildly solubilized and incubated in sodium phosphate buffer with either pH 6.0, 7.0, or 8.0. After separation of soluble and membrane-bound proteins, pellets (P) and supernatants (S) were analyzed by immunoblotting with antibodies against Tic62 and FNR. A typical result of four independent experiments is shown. Furthermore, the amount of Tic62 and FNR in the pellet (gray) and supernatant (white) of all experiments (n = 4) was quantified (including se bars) and is depicted as fraction of total sample (100%). [See online article for color version of this figure.]

Figure 6.

Tic62 Can Bind Both Arabidopsis FNR Isoforms in Vitro, and the Interaction Is Stable under High Salt Concentrations. (A) FNR is more loosely bound to thylakoids from tic62 than from wild-type plants. Isolated thylakoids of wild-type and tic62 plants were washed with high ionic strength buffer (0.5 M NaCl), and membrane and soluble fractions were separated by centrifugation. Shown is a representative immunoblot of supernatant (S) and pellet (P) fractions obtained from wild-type and tic62 thylakoids, using a dilution series of protein and chlorophyll concentrations and probed with FNR antibody. Additionally, the quantification of the FNR amount in wild-type (white; 100%) and tic62 (gray) supernatant and pellet signals from the immunoblots is shown. The dotted line represents the amount of FNR detected in native tic62 thylakoids. Standard error bars are included; the experiment was performed in triplicate. (B) Cyt c reduction activity in the supernatant of high salt washes of tic62 thylakoids is ∼70% of wild-type activity. Activity was determined by Fd-dependent Cyt c reduction, monitored with a spectrophotometer at 550 nm. The experiment was performed in triplicate; se bars are included. (C) Tic62 binds both LFNR1 and LFNR2 equally well. LFNR1/LFNR2 binding assay on Tic62 Ct-His affinity matrix. Overexpressed and purified Tic62 C terminus and FBPase were bound via a (His)₆-tag to Ni²⁺ beads and used as an affinity matrix for LFNR1 and LFNR2 from tic62 Arabidopsis stroma. An empty column without the addition of His-tagged protein was used as additional negative control. After incubation, the matrix was washed (W; last wash), and bound proteins were eluted by addition of 750 mM NaCl (E1), 1 M NaCl (E2), 4 M urea (E3), 8 M urea (E4), 200 mM imidazole (E5), and 400 mM imidazole (E6). The resulting samples including 1/70 of load (L) and flow-through (FT) were subjected to urea/SDS-PAGE and immunoblotting with FNR antibody.

Figure 7.

Tic62 Binds to a Novel Binding Site at the Back Side of FNR. Mapping of chemical shift perturbations caused by the binding of the Tic62-R1 peptide, Fd, and NADP⁺ onto the tertiary structure of FNR. Residues which exhibited chemical shift perturbations upon addition of the factors are shown in red. The FAD binding domain is highlighted in blue and the NADP⁺ binding domain in green. The front side and the back side of the FNR are displayed.

Figure 8.

Tic62 Has a Stabilizing Effect on FNR in Vitro. Catalytic activity of overexpressed and purified FNR was measured in vitro using the Fd-dependent Cyt c reduction assay. FNR was either used fresh (white), or activity was determined after overnight incubation (black). Both values are additionally represented by dotted lines for comparison with the other results. Various amounts (molar ratio of FNR:used protein was 1:2, 1:1, and 1:0.5) of full-length Tic62 (light gray), Tic62 C terminus (gray), or egg albumin as control (dark gray) were added to FNR before overnight incubation, and activity was measured the next day. None of the interacting proteins displayed any detectable Cyt c reductase activity independently of FNR. The mean values of triplicate experiments with se bars are depicted.