Abstract
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.
Highlights
Phosphatase treatment prior to the analysis strongly reduced ProQ-Diamond staining when compared with untreated Toc159A-tag plants (TAP) eluate
To roughly characterize the identified kinase activities, a similar experiment was repeated in the presence of kinase inhibitors: heparin, 5,6-dichlorobenzimidazole riboside (DRB; inhibiting CK2 and cyclin-dependent kinase), apigenin, chelerythrine, or nonradioactive GTP as a potential competitor (CK2; Fig. 6C)
Phosphorylation of Toc159A-His-6x by the SN100 and the P100 fractions was effectively inhibited by heparin at a concentration of 15 mg mL21 (Fig. 6C, lanes 4 and 12)
Summary
To roughly characterize the identified kinase activities, a similar experiment was repeated in the presence of kinase inhibitors: heparin (inhibiting CK2, protein kinase C [PKC], and Ca2+/calmodulin-dependent protein kinase), 5,6-dichlorobenzimidazole riboside (DRB; inhibiting CK2 and cyclin-dependent kinase), apigenin (inhibiting CK2 and mitogen-activated protein kinase), chelerythrine (inhibiting PKC), or nonradioactive GTP as a potential competitor (CK2; Fig. 6C). Phosphorylation of Toc159A-His-6x by the SN100 and the P100 fractions was effectively inhibited by heparin at a concentration of 15 mg mL21 (Fig. 6C, lanes 4 and 12). Cold GTP at 500 mM clearly compete
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