Purification and Characterization of the Preprotein Translocase of the Outer Mitochondrial Membrane from Arabidopsis. Identification of Multiple Forms of TOM20

Abstract

The translocase of the outer mitochondrial membrane (TOM) complex is a preprotein translocase that mediates transport of nuclear-encoded mitochondrial proteins across the outer mitochondrial membrane. Here we report the purification of this protein complex from Arabidopsis. On blue-native gels the Arabidopsis TOM complex runs at 230 kD and can be dissected into subunits of 34, 23, 21, 8, 7, and 6 kD. The identity of four subunits could be determined by immunoblotting and/or direct protein sequencing. The 21-and the 23-kD subunits exhibit significant sequence homology to the TOM20 preprotein receptor from other organisms. Analysis by two-dimensional isoelectric focusing/Tricine sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the presence of further forms for Arabidopsis TOM20. All TOM20 proteins comprise a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria. Clones encoding four different forms of Arabidopsis TOM20 were identified and sequenced. The deduced amino acid sequences are rather conserved in the N-terminal half and in the very C-terminal part, but include a highly variable glycine-rich region close to the C terminus. Implications on the function of plant TOM complexes are discussed. Based on peptide and nucleic acid sequence data, the primary structure for Arabidopsis TOM40 is presented.Prerequisites for protein transport into mitochondria are targeting information of the proteins to be transported and a mitochondrial "protein import apparatus" that decodes the targeting information and mediates translocation of proteins across the organellar membranes (for review, see Neupert, 1997;Mori and Terada 1998; Braun and Schmitz, 1999;Voos et al., 1999). The targeting information often is localized on N-terminal extensions, termed presequences, which are removed within the organelles by processing peptidases (Braun and Schmitz, 1998). Central components of the protein import apparatus are translocase complexes: the preprotein translocase of the outer mitochondrial membrane (the so-called TOM complex) and the two preprotein translocases of the inner mitochondrial membrane (called TIM complexes). These translocases were first described for the fungi yeast and Neurospora crassa (for review, see Meisinger et al

Eukaryotic Peptide Deformylases. Nuclear-Encoded and Chloroplast-Targeted Enzymes in Arabidopsis

“…3C). Significant mitochondrial contamination of the Arabidopsis chloroplast preparation is also unlikely given that mitochondrial preparations from Arabidopsis green tissues are difficult (Werhahn et al, 2001) and mitochondria and chloroplasts are collected from 23% (v/v)/40% (v/v) and 50% (v/v)/70% (v/v) Percoll interfaces, respectively (Rensink et al, 1998;Werhahn et al, 2001).…”

Novel Targeting Signals Mediate the Sorting of Different Isoforms of the Tail-Anchored Membrane Protein Cytochrome b5 to Either Endoplasmic Reticulum or Mitochondria

“…High magnification confocal laser-scanning microscopy (CLSM) revealed that the fluorescence pattern attributable to Cb5-D was actually toroidal in shape ( Figure 2I) and that these structures enclosed the spherical fluorescent structures attributable to endogenous E1b ( Figures 2J and 2K). Coexpression of Cb5-D and green fluorescent protein (GFP)-Tom22 (a fusion protein consisting of GFP and the Arabidopsis thaliana 22-kD subunit of the translocase of the outer mitochondrial membrane; Werhahn et al, 2001) resulted in colocalization of these proteins in the same torus structures ( Figures 2L to 2N), indicating that Cb5-D was localized specifically to the outer membrane of mitochondria. Additional CLSM experiments revealed that the reticular fluorescence patterns attributable to ERlocalized Cb5-A, -B, and -C did not colocalize with, nor enclose, the spherical fluorescent structures attributable to E1b in the mitochondrial matrix (data shown only for myc-Cb5-A; Figures 2O to 2Q).…”

Carbonic Anhydrase Subunits Form a Matrix-exposed Domain Attached to the Membrane Arm of Mitochondrial Complex I in Plants

“…Isolation and Subfractionation of Mitochondria from ArabidopsisMitochondria from Arabidopsis were purified as outlined in Werhahn et al (18). Organelles were resuspended in resuspension buffer (0.4 M mannitol, 1 mM EGTA, 10 mM Tricine, 0.2 mM PMSF, pH 7.2) and either stored at Ϫ80°C or directly used for the generation of mitochondrial subfractions.…”