Expression and functional roles of the two distinct NDH-1 complexes and the carbon acquisition complex NdhD3/NdhF3/CupA/Sll1735 in Synechocystis sp PCC 6803

Abstract

To investigate the (co)expression, interaction, and membrane location of multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes and their involvement in carbon acquisition, cyclic photosystem I, and respiration, we grew the wild type and specific ndh gene knockout mutants of Synechocystis sp PCC 6803 under different CO2 and pH conditions, followed by a proteome analysis of their membrane protein complexes. Typical NDH-1 complexes were represented by NDH-1L (large) and NDH-1M (medium size), located in the thylakoid membrane. The NDH-1L complex, missing from the DeltaNdhD1/D2 mutant, was a prerequisite for photoheterotrophic growth and thus apparently involved in cellular respiration. The amount of NDH-1M and the rate of P700+ rereduction in darkness in the DeltaNdhD1/D2 mutant grown at low CO2 were similar to those in the wild type, whereas in the M55 mutant (DeltaNdhB), lacking both NDH-1L and NDH-1M, the rate of P700+ rereduction was very slow. The NDH-1S (small) complex, localized to the thylakoid membrane and composed of only NdhD3, NdhF3, CupA, and Sll1735, was strongly induced at low CO2 in the wild type as well as in DeltaNdhD1/D2 and M55. In contrast with the wild type and DeltaNdhD1/D2, which show normal CO2 uptake, M55 is unable to take up CO2 even when the NDH-1S complex is present. Conversely, the DeltaNdhD3/D4 mutant, also unable to take up CO2, lacked NDH-1S but exhibited wild-type levels of NDH-1M at low CO2. These results demonstrate that both NDH-1S and NDH-1M are essential for CO2 uptake and that NDH-1M is a functional complex. We also show that the Na+/HCO3- transporter (SbtA complex) is located in the plasma membrane and is strongly induced in the wild type and mutants at low CO2.

Figure 1.

Growth Curves of Wild-Type Synechocystis 6803 Cells and M55, ΔNdhD3, ΔNdhD4, ΔNdhD3/D4 Double Mutant, and ΔNdhD3/D4/SbtA Triple Mutant. (A) Growth in BG-11 medium, pH 7.5, 3% CO₂. (B) Growth in BG-11 medium, pH 7.5, air level of CO₂. (C) Growth in BG-11 medium, pH 8.3, air level of CO₂.

Figure 2.

Membrane Protein Complexes of Synechocystis 6803 Wild Type and the M55 Strain. Wild-type and M55 cells were grown at 3% CO₂ in BG-11 medium at pH 7.5 or at air level of CO₂ in BG-11 medium at pH 8.3. Cells were then harvested and crude thylakoid membranes isolated as described in Methods. After solubilization of membranes with 1.5% n-dodecyl-β-d-maltoside, the protein complexes were separated by BN-PAGE. (A) The BN-gel was stained with silver. Molecular markers are indicated to the left, and the assignment of the major protein complexes is given to the right. (B) Protein complexes were electroblotted to a polyvinylidene difluoride (PVDF) membrane, and the membrane was first probed with anti-NdhJ to identify the NDH-1 complexes (NDH-1L and NDH-1M) and subsequently with anti-NdhD3, which recognized the NDH-1S₁ and NDH-1S₂ complexes. (C) PVDF membrane was first probed with anti-NdhK and subsequently with anti-SbtA to recognize the Na⁺/HCO₃⁻ transporter. Protein bands indicated by arrows interacted with respective antibodies.

Figure 3.

Two-Dimensional Analysis of Synechocystis 6803 Wild Type and M55 Membrane Protein Complexes from Cells Grown at Low CO₂, pH 8.3. After separation of the protein complexes in the BN gel, the lane was cut out, solubilized with Laemmli buffer, and subjected to SDS-PAGE. (A) Silver stained gels of wild-type and M55 membranes (crude thylakoid preparations). (B) Gels were electroblotted to a PVDF membrane and probed sequentially with NdhK, NdhJ, NdhD3, NdhF3, and SbtA antibodies revealing the presence or absence, as well as the positions, of the NDH-1L, NDH-1M, NDH-1S complexes (S₁ and S₂), and the SbtA complex.

Figure 4.

Identification by MALDI-TOF Mass Spectrometry of the NdhK Protein Present in the NDH-1L and NDH-1M Complexes, the CupA Protein Present in the NDH-1S₁ Complex, and the SbtA Protein in the HCO₃⁻ Transporter Complex. Protein spots for identification were taken from the gel in Figure 3. Essentially similar SbtA spectra were obtained from three different partially overlaying spots. m/z, mass-to-charge ratio.

Figure 5.

Sections of Silver-Stained 2-D BN/SDS-PAGE Gels from Synechocystis Wild Type and M55, ΔNdhD3, ΔNdhD4, ΔNdhD3/D4 Double Mutant, and ΔNdhD3/D4/SbtA Triple Mutant, Enclosing the Major Components of the Carbon Acquisition Complexes and the PSII Monomer and Dimer Complexes. Major PSII proteins CP47, CP43, D2, and D1 present in PSII monomers and dimers, identified by MALDI (data not shown), are indicated to the left. Membranes were isolated from cells grown under high CO₂, pH 7.5, and after a CO₂ downshift at pH 7.5 and 8.3 for 24 h. The NdhK protein in NDH-1L and NDH-1M complexes, CupA in the NDH-1S₁ complex, and SbtA are indicated by arrows. CP47 released from the PSII monomer in M55 cells shifted to low CO₂, pH 7.5, was identified with mass spectrometry (data not shown) and is likewise indicated by an arrow.

Figure 6.

Proteomes of the Membrane Protein Complexes of the PSI-Less Mutant and the ΔNdhD1/D2 Mutant as Compared with the Wild-Type Strain Grown under Similar Conditions. (A) The wild type and the PSI-less mutant were grown in BG-11 medium supplemented with 5 mM glucose at low CO₂, pH 7.5, 5 μmol photons m⁻² s⁻¹. The crude thylakoid membrane fraction was isolated and subjected to 2-D BN/SDS-PAGE, and the gel was stained with silver. (B) The wild type and the ΔNdhD1/D2 mutant were grown at low CO₂, pH 7.5, and 50 μmol photons m⁻² s⁻¹. On the top of the silver-stained 2-D gels is shown an immunoblot of one-dimensional BN gel probed with anti-NdhJ to demonstrate the locations and abundances of the NDH-1L and NDH-1M complexes in silver-stained gels below, prepared after 2-D BN/SDS-PAGE. Below wild-type membranes is shown an NdhF1 immunoblot after 2-D BN/SDS-PAGE. Arrows indicate the spot reacting with anti-NdhF1.

Figure 7.

Two-Dimensional Analysis of Membrane Protein Complexes from T. elongatus Cells Grown at High and Low CO₂, pH 8.3. NDH-1L, NDH-1M, and NDH-1S complexes are marked on the top of the gel.

Figure 8.

Location of the Various Carbon Acquisition Systems and NDH-1 Complexes in the Thylakoid Membrane and the Plasma Membrane. The purified membrane fractions were obtained by sucrose density fractionation and subsequent purification of the thylakoid and plasma membranes in the two-phase partitioning system composed of dextran and polyethylene glycol. CP43 and NrtA were used as markers for the purity of the plasma and the thylakoid membrane fractions, respectively. Anti-NdhD3 and anti-NdhF3 were used to localize the marker proteins of the NDH-1S complexes and anti-NdhJ and anti-NdhK the marker proteins of the NDH-1L and NDH-1M complexes to the thylakoid membrane, whereas Anti-SbtA localizes the Na⁺/HCO₃⁻ transporter to the plasma membrane.

Figure 9.

Immunoblots Demonstrating the Accumulation of the NdhD3, NdhF3, NdhF1, NdhK, and SbtA Proteins in the Total Membrane Fractions of Synechocystis Wild Type and Several Ci Acquisition Mutant Strains. The cells were first grown at high CO₂ (lane 1) and then shifted to low CO₂ at pH 7.5 (lane 2) or 8.3 (lane 3) for 24 h before isolation of the total membrane fractions of the cells. To work on the linear region of the immunoresponse with different antibodies, 5 μg of membrane proteins were loaded in the well for detection with SbtA antibody, 20 μg protein for detection with the NdhK, NdhD3, and NdhF3 antibodies, and 40 μg protein for detection with the NdhF1 antibody.