The plastid ndh genes code for an NADH-specific dehydrogenase: isolation of a complex I analogue from pea thylakoid membranes

Abstract

The plastid genomes of several plants contain ndh genes-homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria. From sequence similarities with these genes, the ndh gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established. Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant Pisum sativum. The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step. The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS/PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting. The Ndh complex showed an NADH- and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors. This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase. Our results are compatible with a dual role for the Ndh complex in the chlororespiratory and cyclic photophosphorylation pathways.

Figure 1

Estimation of mitochondrial contamination in preparations of thylakoid membranes. The TM and TP preparations of pea thylakoid membranes and mitochondrial membranes (Mito) were separated by SDS/PAGE and immunoblotted with antibodies specific for cytochrome oxidase subunit 1 (Cox1). The bottom line indicates NADH:FeCN activity corresponding to the amounts of protein loaded on the gel. A stromal lamellae (SL) fraction of thylakoid membranes, which is enriched in NADH:FeCN activity and Ndh proteins content (8, 10) is shown for comparison.

Figure 2

Anion-exchange chromatography. (A Upper) Q-Sepharose HP step. A sample of selectively solubilized thylakoid membranes was applied to the column and eluted with a linear NaCl gradient. All fractions were assayed for NADH:FeCN (•) and NADPH:FeCN (□) activity. Protein concentration was monitored by absorbance at 280 nm (▵). Some of the fractions, known not to contain Ndh proteins from preliminary experiments, were pooled prior to SDS/PAGE. (Lower) Immunoblots of the fractions probed with antibodies specific for NdhK and FNR. (B Upper) Mono Q HR 5/5 step. Peak fractions from Q-Sepharose step (fractions 8 and 9) were diluted, applied to the column, and eluted with a linear NaCl gradient. Fractions were assayed for activity and probed with anti-NdhK antibody (Lower). The NADPH:FeCN activity of all the fractions was less than 3% of the NADH:FeCN activity. The elution profile of the activity [Mito.NADH:FeCN (○)] from solubilized mitochondrial membranes (20 μmol/min of activity applied) under identical conditions of chromatography is shown for comparison.

Figure 3

Size-exclusion chromatography of the Ndh complex. The peak fraction after the Mono Q step (fraction 3) was concentrated and applied to the Sephacryl S-300 HR column. (A) Assay of all fractions containing protein (estimated by absorbance at 280 nm) for NADH:FeCN activity. (B) Immunoblot analysis. The same fractions separated by SDS/PAGE and probed with antibodies specific for NdhK, I, and J. Positions corresponding to the elution of the molecular mass standards are indicated by arrows. (C) Silver-stained SDS/PAGE gel. The coeluting subunits of the Ndh complex, some of which were identified (see text), are indicated on the left. Several impurities of smaller total molecular mass are marked by dots on the right. Positions of molecular mass markers are also indicated on the right.

Figure 4

Comparison between the N-terminal sequences of NdhA, NdhH, and NdhK subunits (Fig. 3C) of purified pea Ndh complex and sequences predicted from the plastid genome of other plants. The sequences of the ndh genes from pea are not known; therefore, the N-terminal data for pea (Pisum sativum) is compared with the predicted sequences of the NdhA, NdhH, and NdhK proteins from tobacco (Nicotiana tabacum) (1) and yellow lupine (Lupinus luteus) (Swiss Prot accession no. P52766). Asterisks, identical residues; dots, conservative changes. Numbers indicate position of the residue from the N terminus.

Figure 5

Possible role of the Ndh complex. Scheme shows the Ndh complex as a component both of cyclic electron flow around photosystem I (PSI) and of a putative chlororespiratory pathway. Fd, ferredoxin; PC, plastocyanin; PQ, plastoquinone; FNR, ferredoxin:NADP⁺ reductase; nH⁺, an unknown number of protons pumped. Reduction of NAD⁺ by NADPH may be catalyzed either directly through a putative transhydrogenase (TH) or indirectly through substrate cycles. Independent cyclic pathway possibly catalyzed by PsaE subunit of PSI is also indicated.