Non-canonical Transit Peptide for Import into the Chloroplast

Abstract

The large majority of plastid proteins are nuclearencoded and, thus, must be imported within these organelles. Unlike most of the outer envelope proteins, targeting of proteins to all other plastid compartments (inner envelope membrane, stroma, and thylakoid) is strictly dependent on the presence of a cleavable transit sequence in the precursor N-terminal region. In this paper, we describe the identification of a new envelope protein component (ceQORH) and demonstrate that its subcellular localization is limited to the inner membrane of the chloroplast envelope. Immunopurification, microsequencing of the natural envelope protein and cloning of the corresponding full-length cDNA demonstrated that this protein is not processed in the N-terminal region during its targeting to the inner envelope membrane. Transient expression experiments in plant cells were performed with truncated forms of the ceQORH protein fused to the green fluorescent protein. These experiments suggest that neither the N-terminal nor the C-terminal are essential for chloroplastic localization of the ceQORH protein. These observations are discussed in the frame of the endosymbiotic theory of chloroplast evolution and suggest that a domain of the ceQORH bacterial ancestor may have evolved so as to exclude the general requirement of an N-terminal plastid transit sequence.Higher plant plastids contain a genome with limited coding capacity. The large majority of plastid proteins are nuclearencoded and thus, must be imported within these organelles. To this purpose, the two envelope membranes that surround chloroplasts contain a protein import apparatus constituted of the TOC and TIC complexes (translocon at the inner or outer membranes of the chloroplast envelope) (for recent reviews, see Refs. 1-4). The major element of this translocation complex is Toc75, which is the most abundant protein in the outer envelope membrane. Moreover, Toc75 seems to form the central pore of the outer envelope translocation channel (5, 6) and interacts specifically with the N-terminal transit peptide of precursor proteins during the import process toward chloroplast (7). This cleavable and N-terminal transit peptide was shown to be necessary and sufficient for transport of precursors (i) across the two envelope membranes (8, 9) and (ii) across the thylakoid membrane if the transit sequence is bipartite (it contains additional targeting information for thylakoid lumen targeting) (9). The cleavage of the transit sequence is performed by two proteases, one in the stroma (10) and one in the thylakoid lumen for the bipartite transit sequences (11). Moreover it is interesting to note that all described inner membrane proteins and intermembrane space proteins possess an N-terminal cleavable transit peptide, while most outer envelope membrane proteins do not have any cleavable transit peptide. In the latter case, the targeting information is contained within the mature protein (12), and after their cytosolic synthesis, proteins are directly incorporated in the lipid bilayer th...

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