Proteomic Approach to Identify Novel Mitochondrial Proteins in Arabidopsis^{<a href="#FN1" rid="FN1" class=" fn">1</a>}

Abstract

Mitochondria play a pivotal role in energy metabolism of eukaryotic cells. Mitochondria are the location of numerous catabolic reactions, many of which are coupled to the reduction of NAD^{, they are the location of the respiratory chain that reoxidizes NAD, transfers electrons to molecular O}₂, and generates a proton gradient across the inner mitochondrial membrane, and they are the site of ADP phosphorylation by the ATP synthase complex. Furthermore, mitochondria are involved in several anabolic reactions: Mitochondria can synthesize amino acids, nucleotides, lipids, and prosthetic groups, such as heme, biotin, and lipoic acid. Mitochondria have their own genetic system and protein biosynthesis machinery. Finally, mitochondria seem to have central regulatory functions for the eukaryotic cell, e.g. in apoptosis (Gottlieb, 2000). To perform all of the addressed functions, mitochondria need a large number of different proteins, most of which are nuclear encoded and post-translationally transported into the organelle (Lithgow, 2000).

Mitochondria from plants have additional functions (Mackenzie and McIntosh 1999; Rasmusson et al., 1999). Plant mitochondria indirectly participate in photosynthesis, because an important step of the photorespiratory pathway—the decarboxylation of Gly—takes place in mitochondria (Raghavendra et al., 1998). Plant mitochondria have special ways for malate oxidation, which are based on the presence of an NAD-dependent malic enzyme (Winning et al., 1994). Plant mitochondria are capable of synthesizing Met, folate, and thymidylate (Neuburger et al., 1996; Rébeillé et al., 1997; Ravanel et al., 1998). The respiratory chain of plant mitochondria is much more branched than in other organisms: There is a cyanide-insensitive respiration, which is based on the alternative oxidase, and there are also alternative NADH dehydrogenases, which can use internal and external NADH or NADPH as substrates (Vanlerberghe and McIntosh, 1997; Rasmusson et al., 1999). The cytochrome c reductase of the respiratory chain of plants is a bifunctional enzyme because it comprises a protease activity that is responsible for the removal of presequences of nuclear-encoded mitochondrial proteins (Braun et al., 1992). The preprotein translocase of the outer mitochondrial membrane (also called the TOM complex) contains fewer preprotein receptors with broader substrate specificity (Jänsch et al., 1998; Braun and Schmitz, 1999; Werhahn et al., 2001). Finally, the genetic system of plant mitochondria is very special (for review, see Brennicke et al., 1999; Mackenzie and McIntosh, 1999). The plant mitochondrial genome is comparatively large, transcripts in plant mitochondria undergo editing before they are translated, and some transcripts are generated by trans-splicing. The genome of plant mitochondria undergoes rearrangements, which can have important implications, e.g. in causing cytoplasmic male sterility (Janska et al., 1998). To identify further functions of plant mitochondria and to better understand their complex role in plant cells, a comprehensive characterization of the plant mitochondrial proteome is necessary.

Recently, proteome analyses became a powerful tool for the investigation of complex cellular processes (for review, see Lottspeich, 1999; Görg et al., 2000) and were also successfully used for genetic and physiological studies in plants (for review, see Thiellement et al., 1999). Most proteomic studies are based on the resolution of protein mixtures by two-dimensional (2D) gel electrophoresis and subsequent identification of the resolved proteins by protein sequencing or mass spectrometry. However, the resolution capacity of 2D gel electrophoresis is still insufficient to monitor entire protein sets of eukaryotic cells. Hence, proteome research often is based on a subset of proteins of eukaryotic cells called “subproteome” (Cordewell et al., 2000; Jung et al., 2000). In plant biology, very successful subproteomic analyses were carried out for the cell wall, the plasma membrane, and the thylakoids (Robertson et al., 1997; Santoni et al., 1998, 2000; Peltier et al., 2000; Prime et al., 2000; van Wijk, 2000). During these investigations, hundreds of proteins were separated, several of which were identified for the first time.

Here, we report the characterization of a new subproteome of Arabidopsis, the mitochondrial proteome. Shortly before the completion of the Arabidopsis genome-sequencing project, we started a systematic approach to separate and identify the protein components of plant mitochondria. Optimization of mitochondrial preparations from green Arabidopsis tissues and Arabidopsis suspension cell cultures are reported. More than 50 mitochondrial proteins could be identified by mass spectrometry, direct protein sequencing, and immunoblotting, several of which were previously not described for plant mitochondria. 2D resolutions of mitochondrial proteins under varying conditions are used to define the total complexity of the mitochondrial proteome from Arabidopsis.

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