Selective Homo- and Heteromer Interactions between the Multiple Organellar RNA Editing Factor (MORF) Proteins in Arabidopsis thaliana

Anja Zehrmann,Barbara Härtel,Franziska Glass,Eszter Bayer-Császár,Toshihiro Obata,Etienne Meyer,Axel Brennicke,Mizuki Takenaka

doi:10.1074/jbc.m114.602086

Abstract

RNA editing in plastids and mitochondria of flowering plants requires pentatricopeptide repeat proteins (PPR proteins) for site recognition and proteins of the multiple organellar RNA editing factor (MORF) family as cofactors. Two MORF proteins, MORF5 and MORF8, are dual-targeted to plastids and mitochondria; two are targeted to plastids, and five are targeted to mitochondria. Pulldown assays from Arabidopsis thaliana tissue culture extracts with the mitochondrial MORF1 and the plastid MORF2 proteins, respectively, both identify the dual-targeted MORF8 protein, showing that these complexes can assemble in the organelles. We have now determined the scope of potential interactions between the various MORF proteins by yeast two-hybrid, in vitro pulldown, and bimolecular fluorescence complementation assays. The resulting MORF-MORF interactome identifies specific heteromeric MORF protein interactions in plastids and in mitochondria. Heteromers are observed for MORF protein combinations affecting a common site, suggesting their functional relevance. Most MORF proteins also undergo homomeric interactions. Submolecular analysis of the MORF1 protein reveals that the MORF-MORF protein connections require the C-terminal region of the central conserved MORF box. This domain has no similarity to known protein modules and may form a novel surface for protein-protein interactions.

Highlights

RNA editing in plant organelles requires at least one of nine multiple organellar RNA editing factor (MORF) proteins
Pulldown assays from Arabidopsis thaliana tissue culture extracts with the mitochondrial MORF1 and the plastid MORF2 proteins, respectively, both identify the dualtargeted MORF8 protein, showing that these complexes can assemble in the organelles
The MORF1 and MORF2 proteins, respectively, were introduced as GS-tagged constructs into the genomes of tissue culture cells (24 –26). These bait proteins were immobilized on beads, and the attached proteins were analyzed by tandem mass spectrometry. Both the mitochondrially located MORF1 and the plastid MORF2 proteins pulled down the dual-targeted MORF8 protein among the prominent co-purifying proteins (Table 1)

Summary

Background

RNA editing in plant organelles requires at least one of nine MORF proteins. Results: MORF proteins connect in specific homo- and heteromeric protein-protein interactions. RNA editing in plastids and mitochondria of flowering plants requires pentatricopeptide repeat proteins (PPR proteins) for site recognition and proteins of the multiple organellar RNA editing factor (MORF) family as cofactors. The resulting MORF-MORF interactome identifies specific heteromeric MORF protein interactions in plastids and in mitochondria. The recently identified group of multiple organellar RNA editing factor (MORF, called RIP) proteins may provide the link between the RNA-binding PPR protein and the protein contributing the enzymatic activity [18, 19]. MORF proteins are required for all RNA editing events in plastids and for many, possibly all, sites in mitochondria of A. thaliana. To investigate potential physical contacts between MORF proteins, we have analyzed all possible MORF to MORF connections and report the homomer and heteromer MORF-MORF interactome

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION