NdhM Subunit Is Required for the Stability and the Function of NAD(P)H Dehydrogenase Complexes Involved in CO2 Uptake in Synechocystis sp. Strain PCC 6803

Zhihui He,Min Xu,Yaozong Wu,Jing Lv,Pengcheng Fu,Hualing Mi

doi:10.1074/jbc.m115.698084

Abstract

The cyanobacterial type I NAD(P)H dehydrogenase (NDH-1) complexes play a crucial role in a variety of bioenergetic reactions such as respiration, CO2 uptake, and cyclic electron transport around photosystem I. Two types of NDH-1 complexes, NDH-1MS and NDH-1MS′, are involved in the CO2 uptake system. However, the composition and function of the complexes still remain largely unknown. Here, we found that deletion of ndhM caused inactivation of NDH-1-dependent cyclic electron transport around photosystem I and abolishment of CO2 uptake, resulting in a lethal phenotype under air CO2 condition. The mutation of NdhM abolished the accumulation of the hydrophilic subunits of the NDH-1, such as NdhH, NdhI, NdhJ, and NdhK, in the thylakoid membrane, resulting in disassembly of NDH-1MS and NDH-1MS′ as well as NDH-1L. In contrast, the accumulation of the hydrophobic subunits was not affected in the absence of NdhM. In the cytoplasm, the NDH-1 subcomplex assembly intermediates including NdhH and NdhK were seriously affected in the ΔndhM mutant but not in the NdhI-deleted mutant ΔndhI. In vitro protein interaction analysis demonstrated that NdhM interacts with NdhK, NdhH, NdhI, and NdhJ but not with other hydrophilic subunits of the NDH-1 complex. These results suggest that NdhM localizes in the hydrophilic subcomplex of NDH-1 complexes as a core subunit and is essential for the function of NDH-1MS and NDH-1MS′ involved in CO2 uptake in Synechocystis sp. strain PCC 6803.

Highlights

Thylakoid membranes of cyanobacteria contain a type I NAD(P)H dehydrogenase (NDH-1)2 complex homologous to complex I (NADH:ubiquinone oxidoreductase) from mitochondria and eubacteria [1, 2]
In vitro protein interaction analysis demonstrated that NdhM interacts with NdhK, NdhH, NdhI, and NdhJ but not with other hydrophilic subunits of the NDH-1 complex. These results suggest that NdhM localizes in the hydrophilic subcomplex of NDH-1 complexes as a core subunit and is essential for the function of NDH-1MS and NDH-1MS؅ involved in CO2 uptake in Synechocystis sp. strain PCC 6803
Based on the results of in vitro protein-protein interaction and the assembly intermediates of NDH-1 subcomplex, we propose models for the localization of NdhM in the NDH-1 complexes required for respiration, cyclic electron transport (CET) around photosystem I (PSI), and CO2 uptake

Summary

Experimental Procedures

Culture Conditions—The glucose-tolerant strain of wildtype (WT) Synechocystis 6803 and mutants ⌬ndhK [36] and M55 [3] were cultured at 30 °C in BG-11 medium [37] buffered with Tris-HCl (5 mM, pH 8.0) and bubbled with 5% (v/v) CO2 in air. A DNA fragment encoding a gentamicin resistance (GenR) cassette was amplified by PCR, creating KpnI and BamHI sites using specific oligonucleotide primers (supplemental Table 1) These three products were ligated into the multiple cloning site of pUC19 (Fig. 1A), which was used to transform the WT cells of Synechocystis 6803 as described by Williams and Szalay [38]. Expression and Purification of Fusion Proteins—For testing the direct interaction of NdhM with Ndh subunits, the fragments containing ndhH, ndhI, ndhJ, ndhK, ndhN, ndhO, and ndhS genes were amplified by PCR and cloned into pET28a, respectively, to form His-tagged fusion protein constructs. The ndhM gene was amplified by PCR with primers listed in supplemental Table 1 and cloned into pGBKT7 vector (Gal DNA binding domain; Clontech) to form the bait construct.

Results

Chlorophyll fluorescence parameters

Discussion