1. Volume 10, Issue 4, Pages 575-589 (April 2017)
TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana 
Nargis Parvin, Chris Carrie, Isabelle Pabst, Antonia Läßer, Debabrata Laha, Melanie V. Paul, Peter Geigenberger, Ralf Heermann, Kirsten Jung, Ute C. Vothknecht, Fatima Chigri 
Molecular Plant 
Volume 10, Issue 4, Pages (April 2017)
DOI: /j.molp
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2. Figure 1
Tom9.2kd Plants Are Viable but Have a Severely Reduced Amount of TOM Complex.
(A) Western blot analysis of protein extracts from two representative homozygous dexamethasone-inducible RNAi gene silencing lines of TOM9.2 (tom9.2kd), kd-5-10 and kd-6-12, and wild-type (Wt) plants grown on 1/2 MS agar plates containing dexamethasone (+Dex) or DMSO (solvent control; −Dex). RNAi induced a severe knockdown of TOM9.2 expression, while levels of porin were not affected.
(B) Plate-grown plants treated with dexamethasone (+Dex) or DMSO (−Dex) were transferred to soil and allowed to grow for another 3 days. No outward changes of phenotype were observed for tom9.2kd plants compared with wild-type.
(C and D) Digitonin-solubilized mitochondria from wild-type (Wt) and tom9.2kd plants (line kd-5-10) were resolved on 5%–16% BN–PAGE. Protein complexes were visualized by immunodecoration using antibodies against TOM40, TOM20, and COXII (C) or by Coomassie blue staining and staining for complex I activity (D).
(E) Oxygen consumption rates in normoxic conditions in the dark were measured on 14-day-old tom9.2kd plants grown in liquid medium with dexamethasone (+Dex) or DMSO (−Dex). Data are presented as mean ± SEM of four biological replicates.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

3. Figure 2
Mitochondria from tom9.2kd Plants Show No Alteration in Protein Import Capacity and Steady-State Levels of Proteins.
(A) In vitro protein import was performed with isolated mitochondria from wild-type and tom9.2kd plants grown in liquid culture in the presence of Dex using 35S-radiolabeled mitochondria (TIM23, pAOX1) and chloroplast (pOE33) precursor proteins. After the import reaction, half of each sample was analyzed directly while the other half was treated with thermolysin. All import reactions were analyzed by SDS–PAGE and phospho-imaging.
(B) In vitro protein import was performed as described in (A) using 35S-radiolabeled mitochondria outer membrane proteins (TOM40, TOM20, TOM9.2). After the import reaction, half of each sample was analyzed directly while the other half was treated with Na2CO3 (pH 11, alkaline extraction). All import reactions were analyzed by SDS–PAGE and phospho-imaging.
(C) Western blot analysis of protein abundance in mitochondria isolated from plants grown in liquid culture in the presence of Dex. Serial dilutions were made and resolved on SDS–PAGE and analyzed by immunodecoration using antibodies against different mitochondrial proteins. A Coomassie-stained gel to confirm equal loading is shown in Supplemental Figure 3C.
(D and E) In vitro import assays with 35S-radiolabeled precursor of AOX1 in (D) the presence and absence of α-TOM40 antibody or (E) the presence and absence of CaM. All reactions were analyzed by SDS–PAGE and phospho-imaging.
TL, one-tenth of the radiolabeled precursors used in the assay; Wt, wild-type; tom9.2kd, knockdown plants for TOM9.2; pAOX1, precursor of AOX1; mAOX, mature protein of AOX1.
Molecular Plant  , DOI: ( /j.molp )
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4. Figure 3
Assembly of TOM40 into the TOM Complex but Not the Interaction between TOM40 and TOM20 Is Impaired in Mitochondria from tom9.2kd Plants.
(A) Mitochondria from wild-type (Wt) and tom9.2kd plants were incubated with 35S-labeled precursor protein of TOM40 for the indicated time periods at 26°C. Mitochondria were re-isolated and analyzed by BN–PAGE and phospho-imaging.
(B) Co-immunoprecipitation was performed using digitonin lysed wild-type mitochondria and TOM40, TOM20, or TOM9.2 antibody. Samples were analyzed by SDS–PAGE and immunodecoration with the same antibodies as well as anti-HSP70 as control.
(C) Co-immunoprecipitation was performed using digitonin lysed tom9.2kd mitochondria and antibody against TOM40. Samples were analyzed as described in (B) using antibodies against TOM40, TOM20, and TOM9.2.
L, load; FT, flow-through; W, wash; E, elution.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

5. Figure 4 TOM9.2 Is a CaM-binding Protein within a Cytosolic CaMBD.
(A) n-Dodecyl β-D-maltoside-solubilized mitochondrial extract was loaded onto CaM-agarose beads in the presence of calcium. After washing the beads thoroughly, bound proteins were eluted by replacing calcium with EGTA/EDTA in the buffer. Samples were resolved on SDS–PAGE and immunodetection was performed with antibodies against TOM9.2 and serine hydroxymethyltransferase (SHMT). L, load; FT, flow-through; W, wash; E1 and E2, elutions.
(B) Crosslinking assays between recombinant full-length TOM9.2 and CaM (upper panel) performed either with or without calcium using the 0-Å crosslinker EDC. MBP (lower panel) was used as a control. SDS gel was silver stained to visualize the protein bands. A crosslink product of the right size is marked by an asterisk.
(C) Crosslinking assays between CaM and different TOM9.2 variants in the presence of calcium. FL, full-length TOM9.2; TOM9.2-CD, TOM9.2 cytosolic domain; TOM9.2-ΔCD, TOM9.2 without the cytosolic domain; TOM9.2-ΔCaMBD, TOM9.2 without the potential CaMBD. Protein bands were visualized by Coomassie staining. Crosslink products of the right size are marked by an asterisk.
(D) SDS–PAGE analysis of crosslinking assays with CaM and a peptide comprising the potential CaMBD of TOM9.2 (G31-K51) either in presence or absence of calcium (upper panel). In a control reaction CaM was replaced by BSA (lower panel). Proteins were visualized by Coomassie staining. A crosslink product of the right size is marked by an asterisk.
(E) SDS–PAGE analysis of crosslinking assays between full-length TOM9.2 and CaM in the presence of increasing amounts of peptide. The molar ratio of peptide:TOM9.2 in the assay is indicated. Proteins were visualized by Coomassie staining. Crosslink products of the correct size are marked by an asterisk.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

6. Figure 5 The CaMBD of TOM9 Is Phylogenetically Conserved.
(A) Prediction of CaM-binding motifs in the TOM9.2 sequence shows a high probability (0–9, where 9 is the highest possible score) between R32 and L46.
(B) Helical wheel projection of the potential CaMBD of TOM9.2 between R32 and T49.
(C) Alignment of TOM9/TOM22 sequences of human, yeast, algae, and plants. Only the region of the CaMBD and the beginning of the conserved TM domain is shown. Identical residues are boxed in black, whereas conserved residues are boxed in gray. Three different described CaM-binding motifs found in the TOM9 sequence are indicated above.
Molecular Plant  , DOI: ( /j.molp )
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7. Figure 6
CaM Impairs Assembly of Newly Imported TOM40 into the 390-kDa TOM Complex.
(A) Wild-type mitochondria were pre-treated with increasing amounts of CaM before incubation with 35S-labeled precursor protein of TOM40. Mitochondria were re-isolated and analyzed by BN–PAGE and phospho-imaging.
(B) Wild-type mitochondria were pre-treated with 5 μM CaM before incubation with 35S-labeled precursor proteins of TOM40, TOM20, and TOM9.2. After the import reaction, half of each reaction was treated with Na2CO3 (pH 11, alkaline extraction) and samples were analyzed by SDS–PAGE and phospho-imaging. TL, one-tenth of the 35S-labeled precursor used in the assay.
(C) Digitonin lysed wild-type mitochondria were incubated with or without 5 μM CaM before co-immunoprecipitation was performed with α-TOM40. Samples were analyzed by SDS–PAGE and immunodecoration with antibodies against TOM40, TOM20, and TOM9.2. L, load; FT, flow-through; W, wash; E, elution.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

8. Figure 7 CaM Interferes with the Interaction between TOM9.2 and TOM20.
(A) Fluorescently labeled cytosolic domain of TOM20 (TOM20-CD) corresponding to 100 nM was titrated with unlabeled TOM9.2-CD (black squares), CaM (red triangles), or MBP (blue diamonds). The change in normalized fluorescence (ΔFnorm in [‰]) is plotted against the concentration of the respective unlabeled ligand. ΔFnorm is obtained by subtracting the mean Fnorm value of unbound ligand (baseline) from all data points. Data presented are means of several independent experiments ± SEM.
(B) Constant amounts of labeled TOM20-CD (67 nM) and unlabeled TOM9.2-CD (10 μM) were titrated with unlabeled CaM (black squares) or unlabeled BSA as a control (red diamonds) in presence of calcium. The change in normalized fluorescence (ΔFnorm in [‰]) is presented as described in (A). Data presented here are means of several independent experiments ± SEM.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions