Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes

Abstract

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and β-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.

FIGURE 1.

Isolation and protein analysis of CP47-His and CP43-His isolated from mutants of Synechocystis 6803 blocked at an early stage of PSII assembly. CP43-His (A) and CP47-His (B) were purified by Ni²⁺ affinity chromatography (supplemental Fig. 1), followed by FPLC size exclusion chromatography. Shown are elution profiles of the FPLC size exclusion runs monitored at 280 nm, with the fractions that were later pooled marked by gray areas (start and stop indicated by arrows). a.u., absorbance units. C, CP43-His and CP47-His samples before (pre-FPLC) and after (post-FPLC) purification by FPLC size exclusion chromatography were analyzed on a BN-8–12% (w/v) polyacrylamide linear gradient gel. Samples corresponding to 0.5 μg of Chl a were loaded per lane. A high molecular weight marker (HMW; GE Healthcare) was used to calibrate the gel. D, Coomassie Blue-stained 18% (w/v) SDS-polyacrylamide gel of the isolated proteins and immunoblot analyses with the indicated antibodies. A low molecular mass marker (GE Healthcare) was used to calibrate the gel. WT thylakoid membranes (0.5 μg of Chl a) and final samples of CP47-His and CP43-His (1 μg of Chl a) and PSII-His (1 μg of Chl a) were loaded on the gels. A minor degradation product of CP43-His is indicated by the asterisk.

FIGURE 2.

CP43 and CP47 absorption spectra at room temperature (A) and 5 K (B). The spectra were normalized to the Chl content in the Q_Y region from 645 to 710 nm assuming 13 Chl molecules in CP43 and 16 Chl molecules in CP47 (3, 5, 40). The insets show an enlargement of the absorption in the Q_Y region. RT, room temperature; a.u., absorbance units.

FIGURE 3.

Comparison of the room temperature absorption spectra of CP43-His and CP47-His isolated from Synechocystis 6803 and CP43 and CP47 isolated from spinach. RT, room temperature; Abs, absorbance; a.u., absorbance units.

FIGURE 4.

Comparison of the low temperature absorption and fluorescence spectra in the Q_Y band region between the CP47 and CP43 inner antenna proteins of Synechocystis 6803 and spinach. Absorption and fluorescence spectra for CP47-His (A) and CP43-His (B) from Synechocystis 6803 and for CP47 and CP43 from spinach were recorded at 77 K and normalized to 1 at the absorption and fluorescence maxima. Second derivatives of absorption spectra (2nd Der Abs) are also shown.