The M Domain of atToc159 Plays an Essential Role in the Import of Proteins into Chloroplasts and Chloroplast Biogenesis

Abstract

Toc159, a protein located in the outer envelope membrane and the cytosol, is an important component of the receptor complex for nuclear-encoded chloroplast proteins. We investigated the molecular mechanism of protein import into chloroplasts by atToc159 using the ppi2 mutant, which has a T-DNA insertion at atToc159, shows an albino phenotype, and does not survive beyond the seedling stage due to a defect in protein import into chloroplasts. First we established that transiently expressing atToc159 in protoplasts obtained from the white leaf tissues of ppi2 plants complements the protein import defect into chloroplasts. Using this transient expression approach and a series of deletion mutants, we demonstrated that the C-terminal membrane-anchored (M) domain is targeted to the chloroplast envelope membrane in ppi2 protoplasts, and is sufficient to complement the defect in protein import. The middle GTPase (G) domain plays an additional critical role in protein import: the atToc159[S/N] and atToc159[D/L]mutants, which have a mutation at the first and second GTP-binding motifs, respectively, do not support protein import into chloroplasts. Leaf cells of transgenic plants expressing the M domain in a ppi2 background contained nearly fully developed chloroplasts with respect to size and density of thylakoid membranes, and displayed about half as much chlorophyll as wild-type cells. In transgenic plants, the isolated M domain localized to the envelope membrane of chloroplasts but not the cytosol. Based on these results, we propose that the M domain is the minimal structure required to support protein import into chloroplasts, while the G domain plays a regulatory role.

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

“…This fluorescence may be due either to unassembled protein or a pool of the protein functional in the cytosol. A similar observation has previously been reported for atToc159 (Bauer et al, 2002) and was confirmed using both biochemical and immunohistochemical methods (Hiltbrunner et al, 2001b;Lee et al, 2003). Those data are consistent with partitioning of the protein between the cytosolic compartment and the outer chloroplast membrane.…”

“…These conserved stretches of amino acids may reflect common functional properties such as the ability to associate with the outer membrane or transit sequence binding and suggest that atToc90 may function in ways similar to atToc159. However, atToc90 lacks the N-terminal A-domain typical of atToc159, -132 and -120 (Figure 1b) supporting the notion that this domain has an auxiliary, yet unidentified role (Bauer et al, 2002;Lee et al, 2003).…”

“…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). Cytosolic atToc159 is directly targeted to the Toc-complex in a homotypic interaction with atToc33 mediated by the G-domains of the two proteins (Hiltbrunner et al, 2001b;Bauer et al, 2002;Smith et al, 2002;Lee et al, 2003;Weibel et al, 2003). These results are supported by the crystal structure of the pea Toc34 homodimer identifying sequence motifs conserved in both atToc33 and atToc159 which are required for dimerization and chloroplast targeting of soluble atToc159 (Sun et al, 2002;Weibel et al, 2003).…”

The gateway to chloroplast: re-defining the function of chloroplast receptor proteins

“…Characterization of the T-DNA insertion mutant of Arabidopsis Toc159 (atToc159), ppi2 mutant, showed that the differentiation of proplastids into chloroplasts is arrested, resulting in an albino phenotype (Bauer et al , 2000 ): in other words, the plant cannot develop photoautotrophically. The M-domain, the function of which is to anchor the protein in the outer membrane and to assemble the TOC core complex, was demonstrated to partially complement the preproteins ' import defect in ppi2 mutant (Lee et al , 2003 ). The accumulation and expression level of photosynthesis-related proteins were drastically decreased.…”

“…As aforementioned, the function of the A-domain of Toc159 is still under investigation, largely due to its dispensable function in chloroplast biogenesis (Lee et al , 2003 ). It is highly improbable that the A-domain has evolved and was conserved throughout evolution without having functional significance.…”