SUMMARYMitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein-protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot-gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re-annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource (https://complexomemap.de/at_mito_lea ves) to deposit the data and to allow straightforward access and custom data analyses.

A classical approach, protein separation by two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was combined with tandem mass spectrometry and up-to-date computer technology to characterize the mitochondrial "protein complex proteome" of Arabidopsis (Arabidopsis thaliana) in so far unrivaled depth. We further developed the novel GelMap software package to annotate and evaluate two-dimensional blue native/sodium dodecyl sulfate gels. The software allows (1) annotation of proteins according to functional and structural correlations (e.g. subunits of a distinct protein complex), (2) assignment of comprehensive protein identification lists to individual gel spots, and thereby (3) selective display of protein complexes of low abundance. In total, 471 distinct proteins were identified by mass spectrometry, several of which form part of at least 35 different mitochondrial protein complexes. To our knowledge, numerous protein complexes were described for the first time (e.g. complexes including pentatricopeptide repeat proteins involved in nucleic acid metabolism). Discovery of further protein complexes within our data set is open to everybody via the public GelMap portal at www.gelmap.de/arabidopsis_mito.

Mitochondrial complex I is the main site for electron transfer to the respiratory chain and generates much of the proton gradient across the inner mitochondrial membrane. Complex I is composed of two arms, which form a conserved L-shape. We report the structures of the intact, 47-subunit mitochondrial complex I from Arabidopsis thaliana and the 51-subunit complex I from the green alga Polytomella sp., both at around 2.9 Å resolution. In both complexes, a heterotrimeric γ-carbonic anhydrase domain is attached to the membrane arm on the matrix side. Two states are resolved in A. thaliana complex I, with different angles between the two arms and different conformations of the ND1 (NADH dehydrogenase subunit 1) loop near the quinol binding site. The angle appears to depend on a bridge domain, which links the peripheral arm to the membrane arm and includes an unusual ferredoxin. We propose that the bridge domain participates in regulating the activity of plant complex I.

The mitochondrial oxidative phosphorylation (OXPHOS) system, which is based on the presence of five protein complexes, is in the very center of cellular ATP production. Complexes I to IV are components of the respiratory electron transport chain that drives proton translocation across the inner mitochondrial membrane. The resulting proton gradient is used by complex V (the ATP synthase complex) for the phosphorylation of ADP. Occurrence of complexes I to V is highly conserved in eukaryotes, with exceptions being restricted to unicellular parasites that take up energy-rich compounds from their hosts. Here we present biochemical evidence that the European mistletoe (Viscum album), an obligate semi-parasite living on branches of trees, has a highly unusual OXPHOS system. V. album mitochondria completely lack complex I and have greatly reduced amounts of complexes II and V. At the same time, the complexes III and IV form remarkably stable respiratory supercomplexes. Furthermore, complexome profiling revealed the presence of 150 kDa complexes that include type II NAD(P)H dehydrogenases and an alternative oxidase. Although the absence of complex I genes in mitochondrial genomes of mistletoe species has recently been reported, this is the first biochemical proof that these genes have not been transferred to the nuclear genome and that this respiratory complex indeed is not assembled. As a consequence, the whole respiratory chain is remodeled. Our results demonstrate that, in the context of parasitism, multicellular life can cope with lack of one of the OXPHOS complexes and give new insights into the life strategy of mistletoe species.

HighlightComplex I dysfunction causes reorganization of cellular respiration and affects metabolic processes in mitochondria, plastids, peroxisomes, and other cellular compartments with drastic consequences for growth and development.