Defining theProtein Complex Proteomeof Plant Mitochondria  

Abstract

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.

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

“…These data were consistent with the reported protein complex proteome of plant mitochondria (23). This confirmed several reported CI subunit-containing protein complexes, including CIϩCIII 2 and the mature CI holoenzyme (10,21) (Fig.…”

“…Peptide mass spectrometry analysis revealed 22 different CI-specific subunits in the CI (ND1, ND7, ND9, 75 kDa, 51 kDa, 39 kDa, 20.9 kDa, 18 kDa, ASHI, B14, B13, B14.7, B16.6, B17.2, CA2, CA3, CAL1, NDU10, NDU12, PDSW, PSST, and TYKY) and 23 different subunits in the CIϩCIII 2 (ND1, ND7, ND9, 75 kDa, 51 kDa, 39 kDa, 20.9 kDa, 18 kDa, ASHI, B14, B13, B14.7, B16.6, B17.2, CA2, CA3, CAL1, NDU10, NDU11, NDU12, PDSW, PSST, and TYKY). All of these subunits are confirmed subunits in the plant CI holoenzyme (3,10,23,31). Interestingly, the CI subunit CA2 was also found in spot 37, which was a visible stained region in our BN gels (Fig.…”

“…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 Arabidopsis Class II Sirtuin Is a Lysine Deacetylase and Interacts with Mitochondrial Energy Metabolism

“…The pellet was resuspended in 75 mL of digitonin solubilization buffer. Blue-native gel electrophoresis and second gel dimension was carried out in a standard dual cooled gel electrophoresis chamber (Hoefer) with a gel dimension of 18 3 16 cm as described in Klodmann et al (2011). Gels were either transferred to nitrocellulose membranes for western-blot analysis or stained with Coomassie colloidal blue.…”