Phosphorylation of the Transit Sequence of Chloroplast Precursor Proteins

Abstract

A protein kinase was located in the cytosol of pea mesophyll cells. The protein kinase phosphorylates, in an ATP-dependent manner, chloroplast-destined precursor proteins but not precursor proteins, which are located to plant mitochondria or plant peroxisomes. The phosphorylation occurs on either serine or threonine residues, depending on the precursor protein used. We demonstrate the specific phosphorylation of the precursor forms of the chloroplast stroma proteins ferredoxin (preFd), small subunit of ribulose-bisphosphate-carboxylase (preSSU), the thylakoid localized light-harvesting chlorophyll a/b-binding protein (preLHCP), and the thylakoid lumen-localized proteins of the oxygen-evolving complex of 23 kDa (preOE23) and 33 kDa (preOE33). In the case of thylakoid lumen proteins which possess bipartite transit sequences, the phosphorylation occurs within the stroma-targeting domain. By using single amino acid substitution within the presequences of preSSU, preOE23, and preOE33, we were able to tentatively identify a consensus motif for the precursor protein protein kinase. This motif is (P/G)X n (R/K)X n (S/ T)X n (S*/T*), were n ‫؍‬ 0 -3 amino acids spacer and S*/T* represents the phosphate acceptor. The precursor protein protein kinase is present only in plant extracts, e.g. wheat germ and pea, but not in a reticulocyte lysate. Protein import experiments into chloroplasts revealed that phosphorylated preSSU binds to the organelles, but dephosphorylation seems required to complete the translocation process and to obtain complete import. These results suggest that a precursor protein protein phosphatase is involved in chloroplast import and represents a so far unidentified component of the import machinery. In contrast to sucrose synthase, a cytosolic marker protein, the precursor protein protein kinase seems to adhere partially to the chloroplast surface. A phosphorylation-dephosphorylation cycle of chloroplast-destined precursor proteins might represent one step, which could lead to a specific sorting and productive translocation in plant cells.Chloroplasts and mitochondria contain their own genome; however, the vast majority of their proteins is encoded in the nucleus and synthesized in the cytosol. In general these proteins carry NH 2 -terminal targeting domains which direct them to the proper organelle in a posttranslational event. Whereas some knowledge about the components and mechanisms which are involved in the recognition and translocation of the precursors on the organellar surface has accumulated (Hirsch et al., 1994;Schnell et al., 1994;Gray and Row, 1995;Soll, 1995), almost nothing is known about the events which take place in the cytosol, after the precursor emerges from the ribosome and before binding to the chloroplast. A loosely folded precursor, however, seems to be a prerequisite for membrane translocation. In all import systems aggregation of highly hydrophobic membrane proteins and premature folding has to be prevented. To attain or maintain such an import-competent, soluble conformation,...

The Chloroplast Protein Import Receptors Toc34 and Toc159 Are Phosphorylated by Distinct Protein Kinases

“…2 The amino acid sequence around Ser-113 does not resemble any other canonical phosphorylation motif found in the PROSITE data base. This indicates that the Toc34 kinase may not belong to a conventional protein kinase class.…”

“…In recent years, evidence has accumulated that protein phosphorylation and protein kinases play an important role in regulation and fine-tuning of protein translocation into various subcellular compartments. Upon translation in the cytosol, chloroplast, but not mitochondrial or peroxisomal, preprotein targeting sequences are phosphorylated by a specific protein kinase (2). Phosphorylated preproteins engage an oligomeric guidance complex consisting of a 14-3-3 protein dimer and a cytosolic Hsp70 (3).…”

AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery

“…Instead, it has been proposed that cytosolic factors may target precursors to the chloroplast surface (Schnell and Blobel, 1993). Indeed, a cytosolic member of the 14-3-3 family of proteins has been identified that binds to phosphorylated transit sequences (Waegemann and Soll, 1996) and thus forms a guidance complex to deliver precursor proteins to the Toc-complex (May and Soll, 2000). Recently, we have discovered that atToc159 also exists in an abundant soluble form, partitioning between the outer chloroplast membrane and the cytosol (Hiltbrunner et al, 2001b).…”

The Apicoplast: A Review of the Derived Plastid of Apicomplexan Parasites

“…Four distinct plant transit peptides features are; (1) phosphorylation and dephosphorylation of specific motifs in transit peptides prior to import (this may explain in part the need for hydroxylated residues that can be phosphorylated) (Waegemann and Soll, 1996;Jarvis and Soll, 2001), (2) the presence of Hsp70 recognition domains that mediate binding of these and other cytosolic proteins on route to the chloroplasts (Ivey et al, 2000), (3) specific interactions of transit peptides with chloroplast-specific membrane lipids, and (4) one or more transit peptide domains forming α-helical structures upon contact with the chloroplast membrane lipids. It has recently been noted that Plasmodium transit peptides also typically contain predicted Hsp70 binding domains, and that transit peptide function is sensitive to alterations at these sites (Foth et al, 2003).…”