Efficient assembly of photosystem II in Chlamydomonas reinhardtii requires Alb3.1p, a homolog of Arabidopsis ALBINO3

Abstract

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.

Figure 1.

BN-PAGE of Membranes from Wild-Type, ac29, ac29-44HA, and cbs3 Cells. Membranes of wild-type (1), ac29 (2), ac29-44HA (3), and cbs3 cells (4) were solubilized and separated by a BN-PAGE (see Methods). Arrows indicate the position of complexes containing PSI-LHCI (I_L), PSI (I), PSII-LHCII (II_L), PSII RCC dimer (II₂), PSII RCC monomer (II₁), ATP synthase (A), Rubisco (R), and monomeric (L₁) and trimeric (L_3a and L_3b) light-harvesting complex proteins of PSII, respectively. The molecular masses of standard protein complexes are given in kD.

Figure 2.

BN-PAGE and Fluorography of Chromophores Bound to Membrane Proteins. (A) Membranes labeled with fluorescent dye from wild-type, ac29, and Δycf9 cells were solubilized and proteins separated by BN-PAGE (top) and 2D-BN/SDS-PAGE (bottom). The position of CP26 is marked by an asterisk; dotted frames mark the position of complexes containing LHCII (Figure 1). The molecular masses of standard protein complexes are given in kD. (B) The 77K fluorescence emission spectra of wild-type (violet), ac29 (red), and Δycf9 (dark gray) cells. Protein complexes corresponding to the emission maxima are labeled in the figure body (PSI and PSI-LHCI; PSII₆₈₆ and PSII₆₉₅; LHCI and LHCII, light-harvesting complexes of PSI and PSII, respectively).

Figure 3.

Thermoluminescence and PAM Fluorometry of Wild-Type and ac29 Cells. (A) Flash number–dependent B-band oscillation of charge recombination in PSII was measured by thermoluminescence of dark-adapted wild-type (closed) and ac29 cells (shaded) of C. reinhardtii (see Methods). (B) Induction and photochemical quenching kinetics of chlorophyll fluorescence (PAM) of wild-type (top) and ac29 (bottom) cells were performed as described in Methods. The minimal (F_o), steady state (F_s), and maximal fluorescence yields (F_m and F_m′ under actinic light, respectively) are indicated.

Figure 4.

Autoradiography of Membrane Proteins Isolated from Radiolabeled Wild-Type and ac29 Cells. Wild-type (lanes 1 to 6) and ac29 (lanes 7 to 12) cells (2 × 10⁶) were pulse radiolabeled with [³⁵S]sulfate in vivo for 5, 10, 15, 45, and 75 min (lanes 1 to 5 and 7 to 11) or pulse labeled for 15 min and chased for additional 60 min (lanes 6 and 12). Proteins were separated by SDS-PAGE. Quantification of D1 and AtpA radiolabeling (rel. Intensities) of lanes 1 to 12 is plotted below the autoradiograph.

Figure 5.

Autoradiography of Pulse-Radiolabeled Proteins from Wild-Type and ac29 Cells. (A) Wild-type and ac29 cells (2 × 10⁶) were pulse radiolabeled with [³⁵S]sulfate in vivo for 15 min. Proteins of the soluble (S) and total membrane (M) phases were separated by SDS-PAGE. (B) Total membranes from wild-type and ac29 cells (described in [A]) were incubated with 100 mM sodium carbonate and centrifuged. Proteins from the supernatant (S) and the membrane phase (M) were separated by SDS-PAGE. Arrows point to radiolabeled D1 and AtpA, respectively. (C) Total membranes from wild-type and ac29 cells (described in [A]) were incubated for 30 min at 25°C in the absence (−) or presence (+) of trypsin (T) and β-dodecylmaltoside (D). Percentages of remaining full-length D1 (%D1) in relation to the controls are given under each lane.

Figure 6.

Autoradiography of Membrane Proteins Isolated from Radiolabeled Wild-Type, ac29, ac29-44HA, and cbs3 Cells. Wild-type, ac29, ac29-44HA, and cbs3 cells (2 × 10⁶) were pulse and pulse-chase labeled with [³⁵S]sulfate in vivo as described (Figure 4). Membranes were separated by 2D-BN/SDS-PAGE. Complexes containing radiolabeled D1 protein are presented in a molecular mass window as indicated in kD for BN-PAGE. D1 radiolabeling after 15-min pulse labeling was quantified across the BN-PAGE window, and each spectrum was normalized to the highest peak. Frames (dotted lines) highlight the position of PSII-assembly complexes [RC; RC* reaction center complexes of PSII including D1 and D2; RC47 reaction center complex of PSII including CP47; RCC(1) and RCC(2), monomeric and dimeric RCC complexes of PSII; RCC-LHCII, RCC plus LHCII complexes; free protein, unassembled protein solubilized from the membrane phase].

Figure 7.

Interaction of Alb3.1p with D1. Membranes from strains containing the HA-tagged Alb3.1 (ac29-1HA, cw15-HA), an unrelated HA-tagged control protein (control-HA), or no HA-tagged protein (untransformed) were solubilized as described in Methods. Immunoprecipitations (IP) were performed using HA antiserum (A) or D1 antiserum (B). Ten percent of the solubilized membranes (top panels) and the immunoprecipitated proteins (bottom panels) were analyzed by immunoblotting (IB) with D1 antisera (A) and HA antisera (B).