HiRIEF Proteomics of Arabidopsis thaliana Col-0 and CIAF1 mutants
The assembly of the mitochondrial Complex I requires the expression of nuclear and mitochondrial located genes, co-factor biosynthesis and the assembly of at least 44 subunits. This requires the involvement of assembly factors that interact with subunits of Complex I but are not part of the final holocomplex. A novel plant specific mitochondrial matrix protein encoded by At1g76060, named COMPLEX I ASSEMBLY FACTOR 1 (CIAF1), containing a conserved LYR domain, was shown to be required for Complex I activity. T-DNA insertion mutants of At1g76060 lack the monomeric Complex I and the Supercomplex I+III, displaying the common Complex I growth deficient phenotype. Assembly of Complex I is stalled at 650 and 800 kDa intermediates in mitochondria isolated from these mutants. Evidence points to CIAF1 playing an essential role in the assembly of the peripheral matrix arm Complex I subunits to form the holoenzyme Complex I. We here performed quantitative proteomics by HiRIEF LC-MS with a TMT10plex isobaric tag labeling strategy comparing the control plant Col-0 and several mutant lines, including the mutant with inactivating insertion in At1g76060 (a.k.a. Ciaf1-1, a.k.a. SALK_143656). Among up-regulated proteins were components required for mitochondrial biogenesis, which points to a mitochondrial retrograde signaling pathway being activated and executed in response to the lack of Complex I.
Sample Processing Protocol
Samples of leaves 8 to 10 of the mature rosette of Arabidopsis thaliana were collected from: i) wildtype (Col-0); ii) Ciaf1-1 (SALK_143656) mutant; iii) mutant 1; iv) mutant 2; and v) mutant 3. The leaves were ground in HEPES SDS lysis buffer, and the extracts were heated and sonicated. After centrifugation at 13000 g, the supernatant was collected and processed through the Filter Aided Sample Prep (FASP) protocol (Wisniewski et al, Nature Methods, 2009). The resulting peptides were then labeled with TMT using biological duplicates of each sample type, thus: 126 - WT; 127N - Ciaf1-1; 127C - mut 1; 128N - WT; 128C - Ciaf1-1; 129N - mut 2; 129C - mut 3; 130N - mut 1; 130C - mut 2; 131 - mut 3. Samples were then pooled into one TMT10plex set, cleaned by strong cation exchange-solid phase extraction and separated by High Resolution Isoelectric Focusing (HiRIEF, Branca et al, Nature Methods, 2014) prior to data-dependent LC-MS/MS analysis on a Q-Exactive Mass Spectrometer.
Data Processing Protocol
All MS/MS spectra were searched by MSGF+/Percolator under the Galaxy platform (https://usegalaxy.org) using a target-decoy strategy. The reference database used was Araport11 (40782 protein entries, 2016-06). We used a precursor mass tolerance of 10 ppm and high resolution setting on MS2 level. Only peptides with fully tryptic termini were allowed. We considered carbamidomethylation on cysteine and TMT-10plex on lysine and N-terminus as fixed modifications; and oxidation of methionine as variable modification. Quantitation of TMT-10plex reporter ions was done using an integration window tolerance of 10 ppm. PSMs and peptides were filtered at 1% FDR (peptide level) and proteins were filtered additionally at 1% FDR (protein level) using the “picked” protein FDR method (Savitski et al, Mol Cell Proteomics, 2015).