Modifications at the A-domain of the chloroplast import receptor Toc159

Abstract

Two families of GTPases, the Toc34 and Toc159 GTPase families, take on the task of preprotein recognition at the translocon at the outer membrane of chloroplasts (TOC translocon). The major Toc159 family members have highly acidic N-terminal domains (A-domains) that are non-essential and so far have escaped functional characterization. But recently, interest in the role of the A-domain has strongly increased. The new data of three independent studies provide evidence that the Toc159 A-domain I) participates in preprotein selectivity, II) has typical features of intrinsically unfolded proteins and III) is highly phosphorylated and possibly released from the rest of the protein by a proteolytic event. This hints to a complex regulation of A-domain function that is important for the maintenance of the preprotein selectivity at the TOC translocons.

Figure 1

The inhibitory effect of heparin on chloroplast import does not involve Toc159A. (A) In vitro import assays with chloroplasts of Arabidopsis thaliana wild-type plants and toc159 mutant plants (ppi2) complemented with TAP-tagged Toc159 lacking the acidic domain. Chloroplasts were preincubated for 20 min at 25°C in the dark in the presence of 15 Mg/ml heparin and 20 micromolar ATP. Then ATP was added to a final concentration of 5 mM and the in vitro translated precursor of the small subunit of Rubisco was added. After 0 min or 15 min of import the reactions were stopped and analyzed by SDS-PAGE and autoradiography. (B) Quantification of the import reactions. The amount of [³⁵S] SSu imported after 15 min of import by untreated wild-type chloroplasts was set to 100%.

Figure 2

Hypothetical model on the states of the Toc159 A-domain. The A-domain may exist in an un-phosphorylated state (bottom). Following phosphorylation by CK2 and another kinase at the chloroplast surface it may enter a hyperphosphorylated state (top). Proteolytic cleavage entails the release of the A-domain from the rest of the receptor (right). The signals that trigger the different states of the A-domain and the functional process that involves the A-domain are so far unknown.