The Acidic A-Domain of Arabidopsis Toc159 Occurs as a Hyperphosphorylated Protein

The translocon at the outer membrane of the chloroplast assists the import of a large class of preproteins with amino-terminal transit sequences. The preprotein receptors Toc159 and Toc33 in Arabidopsis (Arabidopsis thaliana) are specific for the accumulation of abundant photosynthetic proteins. The receptors are homologous GTPases known to be regulated by phosphorylation within their GTP-binding domains. In addition to the central GTP-binding domain, Toc159 has an acidic N-terminal domain (A-domain) and a C-terminal membrane-anchoring domain (M-domain). The A-domain of Toc159 is dispensable for its in vivo activity in Arabidopsis and prone to degradation in pea (Pisum sativum). Therefore, it has been suggested to have a regulatory function. Here, we show that in Arabidopsis, the A-domain is not simply degraded but that it accumulates as a soluble, phosphorylated protein separated from Toc159. However, the physiological relevance of this process is unclear. The data show that the A-domain of Toc159 as well as those of its homologs Toc132 and Toc120 are targets of a casein kinase 2-like activity.The Toc and Tic complexes cooperate to import nuclear-encoded chloroplast preproteins from the cytosol (Jarvis, 2008; Kessler and Schnell, 2009). Initially, incoming preproteins encounter the receptors Toc159 and Toc34 at the chloroplast surface. Both are GTP-binding proteins and share sequence homology in their G-domains. While Toc34 is anchored in the outer membrane by a short hydrophobic C-terminal tail, the triple-domain Toc159 is inserted via a largely hydrophilic 52-kD M-domain. In addition to the G- and M-domains, Toc159 has a large acidic A-domain covering the N-terminal half of the protein. Arabidopsis (Arabidopsis thaliana) encodes two isoforms of Toc34 (Toc33 and Toc34) and four of Toc159 (Toc159, Toc132, Toc120, and Toc90; Jackson-Constan and Keegstra, 2001). The Toc159 isoforms have a similar domain structure, but they differ from each other in length and sequence of their A-domain (Hiltbrunner et al., 2001a). However, Toc90 does not have an acidic domain at all and only consists of the G- and M-domains (Hiltbrunner et al., 2004). It has been demonstrated that the A-domain of AtToc159 and AtToc132 have properties of intrinsically disordered proteins (Hernández Torres et al., 2007; Richardson et al., 2009), suggesting an involvement of the A-domain in transient and multiple protein-protein interactions possibly with the transit peptides of preproteins. Toc34 and Toc159 together with the Toc75 channel constitute the Toc-core complex (Schleiff et al., 2003) and are required for the accumulation of highly abundant photosynthesis-associated proteins in the chloroplast. The Arabidopsis deletion mutants of Toc33 (ppi1; Jarvis et al., 1998) and Toc159 (ppi2; Bauer et al., 2000) have indicative phenotypes of their role in chloroplast biogenesis, respectively pale green and albino. Complementation experiments of the ppi2 mutant have established that the G- and M-domains have essential functions whereas the A-domain is dispensable (Lee et al., 2003; Agne et al., 2009). In preceding studies, possibly influenced by the model organism and experimental tools, Toc159 occurred in different forms. Initially, Toc159 was identified in pea (Pisum sativum) as an 86-kD protein lacking the entire A-domain (Hirsch et al., 1994; Bolter et al., 1998). In addition to its membrane-associated form, Arabidopsis Toc159 has been found as a soluble protein (Hiltbrunner et al., 2001b). However, the function and the fate of the A-domain as well as that of soluble Toc159 remain unknown and a matter of debate.Not only GTP binding and hydrolysis by the Toc GTPases but also phosphorylation is known as a regulatory mechanism of chloroplast protein import at the Toc complex level (Oreb et al., 2008b). First, some precursor proteins, such as the small subunit of Rubisco, may be phosphorylated in their transit sequence by a cytosolic kinase (Martin et al., 2006). Phosphorylation promotes binding to a 14-3-3 protein and cytosolic Hsp70 in the guidance complex that delivers the phosphorylated preprotein to the Toc complex (May and Soll, 2000). Second, both Toc159 and Toc34 are known to be phosphorylated and independently so by distinct kinases, OEK70 and OEK98, respectively (Fulgosi and Soll, 2002). These two kinase activities have been located to the outer envelope membrane, but their molecular identification is still pending. Phosphorylation of the Toc GTPases may occur in the GTP-binding domains (Oreb et al., 2008a). For Toc34, data on the site (Ser-113 in pea and Ser-181 in Arabidopsis) and effects of phosphorylation are available (Jelic et al., 2002, 2003). It imposes a negative regulation on the Toc complex by inhibiting GTP and preprotein binding to Toc34, reducing its ability to bind Toc159 and to assemble into the Toc complex (Oreb et al., 2008a). The in vivo mutational analysis in Arabidopsis indicated that phosphorylation at Toc34 represents a nonessential mechanism (Aronsson et al., 2006; Oreb et al., 2007). Despite the 86-kD proteolytic fragment of Toc159 being a major phosphoprotein in the pea outer chloroplast membrane (Fulgosi and Soll, 2002), little is known of the molecular and regulatory mechanisms of Toc159 phosphorylation. In this study, we report that the A-domain of Toc159 can be purified as a stable fragment. Moreover, it is hyperphosphorylated, hinting at an important and highly regulated functional role. Our data suggest that Toc159 is the target of casein kinase 2 (CK2)-like and membrane-associated kinase activities.