Insights into the Composition and Assembly of the Membrane Arm of Plant Complex I through Analysis of Subcomplexes in Arabidopsis Mutant Lines

Abstract

NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) is the largest complex of the mitochondrial respiratory chain. In eukaryotes, it is composed of more than 40 subunits that are encoded by both the nuclear and mitochondrial genomes. Plant Complex I differs from the enzyme described in other eukaryotes, most notably due to the large number of plant-specific subunits in the membrane arm of the complex. The elucidation of the assembly pathway of Complex I has been a longstanding research aim in cellular biochemistry. We report the study of Arabidopsis mutants in Complex I subunits using a combination of Blue-Native PAGE and immunodetection to identify stable subcomplexes containing Complex I components, along with mass spectrometry analysis of Complex I components in membrane fractions and two-dimensional diagonal Tricine SDS-PAGE to study the composition of the largest subcomplex. Four subcomplexes of the membrane arm of Complex I with apparent molecular masses of 200, 400, 450, and 650 kDa were observed. We propose a working model for the assembly of the membrane arm of Complex I in plants and assign putative roles during the assembly process for two of the subunits studied.NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) 2 is the main entry point for electrons in the mitochondrial respiratory chain. It is the largest and most complicated complex of the respiratory chain. In higher eukaryotes, it is composed of more than 40 subunits. It has a dual genetic origin: 5 to 9 subunits are encoded by the mitochondrial genome, and the others are encoded by the nuclear genome. This complex consists of two parts forming an L-shaped structure: the membrane arm forms the base of the L and is embedded within the inner mitochondrial membrane, while the matrix arm forms the side of the L, bonded at one end of the membrane arm and perpendicularly protruding into the soluble matrix space. The matrix arm contains all the Fe-S clusters and the NADH oxidizing activity, while the membrane arm contains the ubiquinone-binding site. The detailed composition, mechanistic analysis of function, and elucidation of the assembly pathway of Complex I have been long-standing areas of research in a range of different species.The localization of the different subunits within the two arms of mitochondrial Complex I has been intensively investigated in the bovine enzyme. This purified Complex I has been fragmented into three parts: , corresponding to the matrix arm, ␤, corresponding to the membrane arm, and ␣, the matrix arm and some membrane subunits that allow the anchoring of the matrix arm in the inner membrane (1). Complex I assembly has been most intensively investigated using mutants of genes encoding Complex I subunits in Neurospora crassa (2) and human (3, 4). Some mutants lacking one Complex I subunits do not contain a fully assembled Complex I but accumulate stable subcomplexes of Complex I. The formation of stable assembly subcomplexes can be identified using antibodies raised against specific Complex I subunits (2-4). Other a...

Subcomplexes of Ancestral Respiratory Complex I Subunits Rapidly Turn Over in Vivo as Productive Assembly Intermediates in Arabidopsis*

“…1). CI subunits have previously been observed at 650 kDa (22), at 400 -450 kDa (3,22,23), and at 200 kDa (3,22,23) in Arabidopsis mitochondrial BN gel analyses. Analysis of gel regions at these native molecular masses and at masses of the complex I subunits observed in the CI holoenzyme and CIϩCIII 2 supercomplex was then carried out.…”

“…In this process, mammalian assembly factors not conserved in other species have been identified (13,14,19). Subcomplexes containing CA2 have been reported using antibodies (11) in detergent disassembly studies of CI in plants (21), in Arabidopsis CI subunit knock-out mutants (22), and in wild type Arabidopsis mitochondrial proteomes in saturation mapping of two-dimensional blue native (BN) gels (23). Knockouts of CA2 and CA3 do not yield a clear phenotype in Arabidopsis (5,11), which might be a consequence of functional redundancy in activities between these proteins.…”

“…The ␥-CAL and 20.9-kDa proteins differed with respect to the subcomplexes into which they progressed, compared with the ␥-CA proteins. Overall, these results demonstrated that the CA domain subunits were not simply added as accessory components to fully or nearly fully assembled CI, but to different degrees they were present in the full range of subcomplexes that are known to include components of the membrane arm of CI (3,22). Import of the ⑀ subunit of ATP synthase, which fails to assemble into a protein complex, was used as a radiolabeled control import for comparison of labeling on gel images.…”

“…Assembly-A range of knock-out mutants for CI subunits leading to the accumulation of certain intermediates has previously been exploited to propose that CA2 is incorporated at the early stage of CI assembly (22), and ␥-CAcontaining subcomplexes have been detected in BN-polyacrylamide gels of wild type mitochondria (23). Using a 15 N labeling approach, we have shown the higher 15 N incorporation rate of subunits contained in subcomplexes than in CI and CIϩCIII 2 , revealing that these structures were not created in vitro by solubilization or electrophoresis artifacts but rather are in vivo intermediates of disassembly or assembly of CI.…”

“…The specific matrix-exposed arm components, including CA2, CAL1, and 20.9 kDa, and mitochondrion-encoded subunits, including ND1, ND7, and ND9, had relatively high NA/(NA ϩ H) ratios and thus lower turnover rates. The plant 20.9-kDa subunit, which has been found in both matrix and membrane modules of CI (3,22), does not have a direct homolog in mammals but is related to fungal NUXM subunits and the 13-and 21-kDa CI subunits in other eukaryotes and was the most stable protein subunit studied in both CI and CIϩCIII 2 .…”