Multistep assembly of chloroplast NADH dehydrogenase-like subcomplex A requires several nucleus-encoded proteins, including CRR41 and CRR42, in Arabidopsis

Abstract

Chloroplast NADH dehydrogenase-like complex (NDH) mediates photosystem I cyclic electron transport and chlororespiration in thylakoids. Recently, substantial progress has been made in understanding the structure of NDH, but our knowledge of its assembly has been limited. In this study, a series of interactive proteomic analyses identified several stroma-localized factors required for the assembly of a stroma-protruding arm of NDH (subcomplex A). In addition to further characterization of the previously identified CHLORORESPIRATORY REDUCTION1 (CRR1), CRR6, and CRR7, two novel stromal proteins, CRR41 and CRR42, were discovered. Arabidopsis thaliana mutants lacking these proteins are specifically defective in the accumulation of subcomplex A. A total of 10 mutants lacking subcomplex A, including crr27/cpn60β4, which is specifically defective in the folding of NdhH, and four mutants lacking NdhL-NdhO subunits, were extensively characterized. We propose a model for subcomplex A assembly: CRR41, NdhO, and native NdhH, as well as unknown factors, are first assembled to form an NDH subcomplex A assembly intermediate (NAI500). Subsequently, NdhJ, NdhM, NdhK, and NdhI are incorporated into NAI500 to form NAI400. CRR1, CRR6, and CRR42 are involved in this process. CRR7 is likely to be involved in the final step, in which the fully assembled NAI, including NdhN, is inserted into thylakoids.

Figure 1.

Characterization of crr41 and crr42 Mutants. (A) Schematic presentation of CRR41 and CRR42 genes and T-DNA insertion sites. White boxes, gray boxes, and a black line indicate untranslated regions, exons, and intron, respectively. (B) Determination of NDH activity using chlorophyll fluorescence. The bottom curve shows a typical trace of chlorophyll fluorescence in wild-type (WT) plants. Leaves were exposed to actinic light (AL; 50 μmol photons m^–2 s^–1) for 5 min. After illumination, the subsequent transient increase in fluorescence ascribed to NDH activity was monitored. Fluorescence levels were normalized against Fm (maximum fluorescence at closed PSII center in the dark) levels. Insets are magnified traces from the boxed area. crr41+CRR41 and crr42+CRR42 represent crr41 and crr42 transformed with CRR41 and CRR42 cDNAs, respectively, expressed under the control of the CaMV 35S promoter. crr41+CRR41-HA and crr42+CRR42-HA: crr41 and crr42 complemented by the introduction of CRR41 and CRR42 cDNA, respectively, fused to the sequence encoding the HA tag under the control of the CaMV 35S promoter. Fo, minimum fluorescence at open PSII center in the dark; ML, measuring light; SP, saturating light pulse.

Figure 2.

Analysis of the NDH Complex in Different NDH Mutant Backgrounds. (A) BN-PAGE analysis of thylakoid protein complexes isolated from wild-type (WT) plants and the indicated mutants. Magnification of the top part of the BN-PAGE gel in Supplemental Figure 1 is shown. After electrophoresis, the gel was stained with Coomassie blue. I, NDH-PSI supercomplex detected in the wild type; II, sub-NDH-PSI supercomplex lacking subcomplex A. (B) Immunoblot analysis of thylakoid proteins, with the indicated antibodies. Thylakoid proteins were loaded on an equal chlorophyll basis, and the series of dilutions is indicated. An asterisk indicates nonspecific signals.

Figure 3.

Immunodetection of CRR41 and CRR42. Freshly isolated chloroplasts from various genetic backgrounds were further fractionated into membrane and stromal fractions. Immunoblot analysis was performed with antibodies against CRR41 (A) and HA tag (B). Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RbcL) and D1 were detected as loading and fractionation controls, respectively. Stromal proteins were loaded by equal protein content, and the series of dilutions is indicated. crr42+CRR42-HA: crr42 transformed with CRR42 cDNA fused to the sequence encoding the HA tag expressed under the control of the CaMV 35S promoter. WT, wild type.

Figure 4.

Accumulation of Stroma-Localized NDH Subunits and Nonsubunit Factors. Stromal proteins were isolated from the wild type (WT) and various mutants, and protein blot analyses were performed with the indicated antibodies. Stromal proteins were loaded by equal protein content, and RbcL was detected as the loading control.

Figure 5.

Formation of NAIs in the Chloroplast Stroma of Various Mutants. Stromal protein complexes isolated from the wild type (WT) crr42 overexpressing the CRR42-HA protein (crr42com) (A), crr41, crr27-1, and ndho (B), crr1-1 and ndhm (C), and crr6 (D) plants were separated by CN-PAGE followed by 2D SDS-PAGE. Proteins were immunodetected with three antibodies against Cpn60α, RbcL, and NdhH or the specific antibodies indicated. Dashed lines indicate the positions of NAIs.

Figure 6.

A Schematic Model of the Chloroplast NDH Subcomplex A Assembly. The nature of NAI800 is unclear. This complex appears to deliver nonnative NdhH to the chaperonin complex containing Cpn60β4 for subsequent protein folding. NdhO, CRR41, and native NdhH, as well as some unknown factors, are first assembled together to form NAI500. Subsequently, NdhJ, NdhK, NdhM, and NdhI are assembled to form NAI400, which accepts NdhN in the final assembly step and then, together with the membrane subunit NdhL and the catalytic subcomplex including NdhS–NdhT, is incorporated into the thylakoids to form the functional NDH-PSI supercomplex. This process is facilitated by CRR41, CRR1, CRR6, HCF101, CRR42, and CRR7 at the various assembly steps indicated. On the basis of their molecular masses, the NAI complexes are expected to contain additional unknown factors that are not depicted in the model. Nonsubunit factors, nucleus-encoded NDH subunits, and chloroplast-encoded NDH subunits are shown in pink, red, and green fonts, respectively.