Unraveling the Functions of Type II-Prohibitins in Arabidopsis Mitochondria. Plant Mol

“Unraveling the functions of type II-prohibitins in Arabidopsis mitochondria.” Plant Mol Biol, J Piechota, M Bereza, A Sokołowska, K Suszyński, K Lech, H Jańska, Department of Biotechnology, University of Wroclaw, 50-383 Wroclaw, Poland, correspondence to:

Supplemental Fig. 1Selection of phb2 and phb6 mutants and phb2 phb6 double mutant. (a) The presence of T-DNA inserts in AtPHB2 and AtPHB6 genes confirmed by PCRs with primers specific to genomic sequences and T-DNA insertions. The PCR reactions contained three primers: LP, RP and LB2 (AtPHB2 gene) or LP, RP and Lbb1 (AtPHB6 gene) (b) The absence of full-length AtPHB2 or AtPHB6 transcripts in phb2 and phb6 mutants confirmed by PCRs with primers amplifying open reading frames of AtPHB2 and AtPHB6 genes. (c) Schemes of AtPHB2 and AtPHB6 genes with indicated T-DNA inserts. Light gray boxes indicate untranslated regions (UTRs) and dark gray boxes represent coding sequences. Arrows indicate the positions of LP, RP, Lbb1 and LB2 primers used in genotyping of phb2 and phb6 plants. (d) The presence of T-DNA inserts in both AtPHB2 and AtPHB6 genes of phb2 phb6 double mutant. (e) The absence of full-length AtPHB2 and AtPHB6 transcripts in phb2 phb6 mutant. Wt – wild type plants.

Supplemental Fig. 2 Comparison between the data obtained from the iTRAQ and Western blot analyzes. The steady-state levels of 12 proteins identified in the proteomes of the wild type (Wt) and phb2 phb6 mitochondria were assayed using Western blot technique. Quantitative data from Western blots are presented as mean ± S.E. from at least three independent biological repeats. The iTRAQ column indicates the phb2 phb6 vs. Wt ratios of the steady-state levels of the proteins assayed using iTRAQ technique. In four cases, antibodies recognized two or more protein isoforms, separately identified using mass spectrometry technique.

Supplemental Fig. 3 Assay for the purity of the mitochondrial fractions. Total leaf extract (TLE) and mitochondrial fraction (Mito) were separated in SDS-PAGE, blotted into PVDF membrane and Western-blotted using antibodies directed against protein representing various cellular compartments: AtVDAC1 (mitochondria), AtHsp70chloro (plastid stroma), AtPsbA (thylakoids), AtH+ATPase (plasma membrane), AtHsp70cyto (cytoplasm).

Supplemental Fig. 4 Relative steady-state levels of proteins downregulated in the phb2phb6 mitochondria and transcripts encoding them. Twenty-three proteins showing the highest decreases in the phb2phb6 mitochondria were selected for analysis. Results are demonstrated as ratios between phb2phb6 mutant vs. wild type plants. Asterisks indicate transcripts with ratios significantly different from 1 (p-value < 0.05). Roman numeral in brackets presented along with gene ID numbers indicate the category to which given protein was assigned according to Table 5: (I) Proteins linked to lipoic acid; (II) Proteins linked to protein quality control system; (III) Proteins linked to electron transport chain.

Supplemental Fig. 5 Pull-down of the 6 × His-tagged AtPHB1 and AtPHB4 proteins. (a) Expression and import of 6 × His-tagged AtPHB1 and AtPHB4 proteins into Arabidopsis mitochondria was confirmed by Western blot analysis of mitochondrial fractions obtained from PHB1-HT, PHB4-HT and wild type (Wt) plants. AtVDAC1 was used as a loading control. (b) Exemplary Western blot showing specific pull-down of His-tagged AtPHB4. Input lanes indicate lysates of PHB4-HT and Wt mitochondria loaded onto HisPur Cobalt Resin. Output lanes represent eluates from HisPur Cobalt Resin. Performed controls (pull-down using Wt mitochondria and Western blot using unrelated AtVDAC1 protein) confirm the specific binding of the AtPHB4-HT to the resin.