Figure 1.
Fractionation of Stt7 and Chlorophyll-Protein Complexes by Sucrose Density Gradient Centrifugation
(A) Thylakoid membranes from Stt7-HA cells in state 1 and state 2 were solubilized with n-dodecyl-β-maltoside, and the chlorophyll-protein complexes were separated by centrifugation on a sucrose density gradient. Proteins from the fractions of the gradients corresponding to Stt7-HA were separated by SDS-PAGE. Immunoblotting was performed with the indicated antibodies; α-P-Thr, anti–phospho-Thr. The identity of the phosphorylated proteins in the lower panel was determined by immunoblotting with antisera from the indicated proteins. In the immunoblot with a-P-Thr, signals corresponding to CP29 and Lhcbm5 are framed.
(B) Immunoblots prepared as in (A) except that the thylakoid membranes from stt7 were used. P11/13 and P17 are the major LHCII proteins of C. reinhardtii and correspond to Type I and Type III LHCII proteins, respectively.
Figure 2.
Levels of Stt7 Decrease under Prolonged State 1 Conditions and High Light
Cells were grown for 120 min under state 2 conditions and subsequently maintained in state 2 (dark + anaerobiosis) or subjected to state 1 conditions (light + strong aeration) or to high light (900 μmol photons m−2 s−1) for 240 min. At various time intervals, total proteins were extracted and the amount of Stt7-HA was measured by immunoblotting with HA antiserum. Cytf was used as a loading control. Top: outline of the experimental conditions (for details, see Materials and Methods).
(A) Cells maintained under continuous state 2 conditions.
(B) Same as (A), but with addition of cycloheximide after the initial culture was maintained in state 2 for 120 min.
(C) Cells in state 2 were subjected to state 1 conditions after 120 min.
(D) Same as (C), but with addition of cycloheximide after 120 min.
(E) Same as (C) but with addition of protease inhibitors after 120 min.
(F) Cells in state 2 were subjected to high light. The levels of PSII and PSI were assessed by immunoblotting with antibodies against D1 and PsaA, respectively.
(G) Same as (F), but with addition of leupeptin. Total proteins were examined by immunoblotting with HA and Cytf antibodies.
(H) Cells shifted to state 1 conditions as shown in (C) were tested for transition from state 1 to state 2 at different times. In this case, transition to state 2 was induced by adding 5 μM FCCP and assayed after 15 min by measuring low-temperature fluorescence emission spectra for each time point.
(I) Stt7 levels of the cells used in (H) were determined by immunoblotting with HA antiserum.
Figure 3.
Coimmunoprecipitations of Stt7 with LHCII, the Cytochrome b6f Complex, and PSI
(A) Right panel: thylakoid membranes from the stt7 mutant strain complemented with Stt7-HA and wild-type (WT) cells in state 1 and state 2 were solubilized with n-dodecyl-β-maltoside, and proteins were coimmunoprecipitated (CoIP) with HA antibodies. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with the antibodies indicated. Left panel: the inputs are shown and represent 10% of the samples used for the coimmunoprecipitations.
(B) Thylakoid membranes from the Stt7-HA or wild-type (WT) strain were processed as above and coimmunoprecipitated with PsaA, Cytf, D1, and AtpA antibodies. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with HA antibodies.
Figure 4.
Stt7 Cofractionates with the Cytochrome b6f Complex
(A) Thylakoid membranes from strains containing Stt7-HA and Stt7-HA with His-tagged Cytf (PetA-His) were solubilized with n-dodecyl-β-maltoside and fractionated by Ni-NTA chromatography. E1, E2 eluates (300 mM imidazole) were analyzed by PAGE and immunoblotting with the indicated antibodies. FT, flow-through; L, loading (10 mM imidazole); W1–W5, washes (20 mM imidazole).
(B) Stt7 interacts with the Rieske protein. GST and GST-Stt7 fusion protein were incubated separately with purified solubilized cytochrome b6f complex from wild-type cells. The eluates (E) (100 mM DTT, 2% SDS) were analyzed by PAGE and immunoblotting with Rieske and Cytf antibodies. FT, flow-through; I, input; W1–W4, washes (PBS).
Figure 5.
Stt7 Contains a Transmembrane Region with Its N-Terminal End in the Thylakoid Lumen
(A) Thylakoid membranes from a strain containing FLAG-Stt7-HA were subjected to increasing concentrations of V8 protease at room temperature, subjected to PAGE, and immunoblotted with HA, FLAG, PsaD, and OEE2 antibodies. An asterisk (*) indicates degradation products of Stt7 revealed with the FLAG antiserum.
(B) Split-ubiquitin assays were performed with fusions of the C-terminal half (Cub) of ubiquitin with either the N- or C-terminal end of Stt7 and the ER protein Alg5 fused at its C-terminal end with the N-terminal half of ubiquitin (Nub). Yeast colonies were plated on permissive (-L-W) and selective (-Ade-H-L-W) media with 10-fold and two 5-fold serial dilutions, respectively.
Figure 6.
Analysis of Mutants of Stt7 Affected in the N-Terminal Region
Transformants of stt7 containing mutant forms of Stt7-HA with changes of Cys68 and Cys73 to Ala/Ser or with a FLAG-tag (FLAG-Stt7-HA) at the N-terminal end were used.
(A) Cys68 and Cys73 of Stt7 are essential for state transitions and LHCII phosphorylation. Fluorescence emission spectra at 70 K of wild type, stt7, and the different transformants under state 1 and state 2 conditions. Right: immunoblots of total extracts from these strains and Stt7-HA with HA antibodies.
(B) Cys68 and Cys73 are required for LHCII phosphorylation under state 2 conditions. Membrane proteins from the wild type and the indicated transformants under state 1 and state 2 conditions were fractionated by SDS PAGE and immunoblotted with an anti–P-Thr antiserum. The bands missing in the transformants are indicated by two arrows.
(C) Insertion of a FLAG tag at the N-terminal end of Stt7 specifically prevents its interaction with the Rieske protein. Thylakoids from Stt7-HA, FLAG-Stt7-HA, and Stt7-C68S were solubilized with n-dodecyl-β-maltoside and immunoprecipitated with HA antiserum. The immunoprecipitate was fractionated by PAGE and immunoblotted with the indicated antibodies.
Figure 7.
Model of Stt7 Kinase within the Thylakoid Membrane in Wild Type, Stt7-C68S, and FLAG-Stt7-HA
The N-terminal part of Stt7 interacts with the lumenal domain of the Rieske protein. A disulfide bridge between Cys68 and Cys73 may be required for the activity of Stt7 that is abolished in Stt7-C68S. Interaction of Stt7 with Rieske protein is prevented by the insertion of a FLAG-tag at the N-terminal end of Stt7. The catalytic domain of Stt7 is on the stromal side of the thylakoid membrane where it could phosphorylate LHCII. The HA-tag of the Stt7-HA is at is C-terminal end on the stromal side of the thylakoid membrane. Fd, ferredoxin; PQH2, plastoquinol.