Transmission electron microscopy of Synechocystis 6803 cells. A, wild-type cells grown aerobically under medium light conditions. B, gun4 mutant cells grown aerobically under medium light intensity. C, gun4 mutant cells grown aerobically under low light intensity. D, gun4 mutant cells grown microaerobically under medium light conditions. Scale bar = 500 nm.

Inactivation of the gun4 gene in Synechocystis 6803 (GT strain). Wild-type and mutant gene copies were amplified using total chromosomal DNA from wild-type and gun4^– strains as templates. PCR amplification of the gun4 gene yielded a correct 1.1-kilobase pair product in the wild type. The mutant gun4 copy gave rise to a larger PCR fragment of 2.6 kilobase pairs due to the insertion of the kanamycin resistance cassette (1.5 kilobase pairs). The sizes of the DNA marker fragments are indicated on the left.

Semiquantitative immunoblot analysis of PSII and PSI subunits in wild type and gun4 mutant. Wild-type and gun4 mutant membrane fractions were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Different amounts of Chl corresponding to designated amounts of cells were loaded per each lane. Immunodetection was performed using polyclonal antibodies raised against the PSII subunits CP47, CP43, and D1, and the PSI subunit PsaD. ATPase band stained on the membrane with Ponceau red indicates protein loading.

Localization, quantification, and activities of both chelatases. A, subcellular localization of Gun4, ferrochelatase (FeCH), and the magnesium chelatase ChlH and ChlD subunits. Membranes have been washed 2 (m2) or 3 (m3) times in thylakoid buffer followed by an additional wash with 2 m NaCl in thylakoid buffer (ms). Membrane fractions (m), crude (c), and soluble extracts (s) of wild-type and gun4 mutant cells were separated by SDS-PAGE and transferred to nitrocellulose membranes. The amount of Chl corresponding to 150 μl of cells at A₇₃₀ = 1 was loaded per each lane. Polyclonal antibodies used for the immunoblots are shown on the right. B, accumulation of the ChlH subunit of magnesium chelatase and ferrochelatase in the crude cell extract were determined by immunodetection. Numbers indicate relative protein loading based on the amount of cells. C, activities of magnesium and ferrochelatase measured in wild-type and gun4 mutant cells.

Analysis of membrane protein complexes and their assembly in wild-type and gun4 mutant cells. A, membrane protein complexes from wild-type and gun4 mutant labeled in vivo with ³⁵[S]Met/Cys were separated in 4–16% BN gel as described under “Experimental Procedures.” Amounts of Chl corresponding to designated amounts of cells were loaded per each lane. B, two-dimensional analysis of membrane protein complexes separated in A. Equal amounts of Chl corresponding to the half (50% A₇₅₀) and double (200% A₇₅₀) amounts of wild-type and gun4^– cells were loaded, respectively. Lanes from the BN-gel were excised and placed on top of the 12–20% denaturating gel. The two-dimensional gels were either stained by Coomassie Blue (Coomassie gels), dried, and exposed to phosphorimaging plates (Autoradiograms) or electroblotted onto a polyvinylidene difluoride membrane that was immunodecorated using antibodies raised against D1 and CP47 proteins (blots). Designation of complexes: RCCI(3) and RCCI(1), trimeric and monomeric PSI complexes, respectively; RCCII(2) and RCCII(1), dimeric and monomeric PSII core complexes, respectively; RC47, the monomeric PSII core complex lacking CP43; RC* and RCa, reaction center complexes accumulating in PSII mutants lacking CP47 (24); U.P., unassembled proteins; iD1 designates D1 processing intermediate.

Phenotype of gun4 mutant cells and affinity purification of Gun4. A, absorption spectra of Synechocystis 6803 wild-type (WT) and mutant cells lacking Gun4 (gun4^–) or expressing a FLAG-tagged version of Gun4 (Δgun4:gun4FLAG). Cells were grown with 5 mm glucose in the medium. Peaks at 620 and 682 nm represent phycocyanin and Chl absorption, respectively. Spectra of whole cells were measured with a UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). B, semiquantitative 77 K fluorescence emission spectra of thylakoid membranes from Synechocystis 6803 wild-type and mutant cells lacking Gun4 (Δgun4) or CP47 (ΔpsbB). Rhodamine was used as an internal standard, and the spectra were normalized to indicate Chl fluorescence per cell. Peaks at 685/695 and 722 nm represent PSII and PSI complexes, respectively. a.u., absorbance units. C, affinity purification of 3×FLAG-Gun4 and identification of magnesium and ferrochelatase (FeCH) subunits by immunodetection. Solubilized extracts of gun4 mutant cells engineered to express 3×FLAG-Gun4 were immunoprecipitated with anti-FLAG M2-agarose (Sigma-Aldrich). FLAG-Gun4 complexes were eluted with 3×FLAG peptide, resolved by SDS-PAGE, stained with Coomassie (D) or blotted onto a nitrocellulose membrane and immunodetected (C). Wild-type eluate was used as a control.