Functional Definition of Outer Membrane Proteins Involved in Preprotein Import into Mitochondria

Abstract

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of β-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.

Plant mitochondria contain the protein translocase subunits TatB and TatC

“…Mitochondria for in vitro import and assays and western blot analysis were harvested from 14-day-old Arabidopsis thaliana seedlings grown in liquid culture as previously described (Lister et al, 2007). Typically, between 2 and 4 mg of mitochondrial protein was obtained from each preparation.…”

“…[ 35 S]Met-labeled precursor proteins were synthesized using Flexi Rabbit Reticulocyte Lysate (Promega) as previously outlined (Chang et al, 2014). In vitro mitochondrial imports were then performed using isolated mitochondria as previously described (Whelan et al, 1995;Lister et al, 2007). All in vitro imports were obtained using radiography and images were scanned using a Typhoon scanner (GE Healthcare).…”

Refining the Definition of Plant Mitochondrial Presequences through Analysis of Sorting Signals, N-Terminal Modifications, and Cleavage Motifs

“…This model accounts for the ability of a single Tom20 in yeast to bind to a diverse array of presequences. Although plants contain a protein that is called Tom20 and that has a receptor function in mitochondrial import, it is not orthologous to yeast or mammalian Tom20 (Perry et al, 2006;Lister et al, 2007). However, the NMR structure of plant Tom20 reveals a similar hydrophobic binding pocket.…”

“…In addition to the fact that plant Tom20s are not orthologous to other Tom20s, plant mitochondria also lack the other two receptor components that have been functionally characterized in yeast, namely Tom70 and Tom22 (Lister et al, 2007). Furthermore, mitochondrial and plastid targeting signals contain significant similarities in plants; thus, plant mitochondrial presequences have evolved to differentiate from the large number and abundant nature of plastid proteins requiring import from the cytosol (Macasev et al, 2000).…”

The gateway to chloroplast: re-defining the function of chloroplast receptor proteins

“…In plants, apart from Toc64, TPR-mediated preprotein translocation has thus far been reported for the mitochondrial outer envelope protein, OM64 and recently, the endoplasmic reticulum (ER) TPR-docking protein, AtTPR7. Both proteins have been shown to interact with chaperones and preproteins Lister et al , 2007 ;Schweiger et al , 2012 ). Intriguingly, both OM64 and AtTPR7 can functionally complement yeast mitochondrial Δ tom70 and Δ sec71 , respectively, thus hinting at a potential function in post-translational protein translocation across the mitochondria and ER membrane.…”