The M domain of atToc159 plays an essential role in the import of proteins into chloroplasts and chloroplast biogenesis.
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.
Lee
Kwang Hee
KH
Center for Plant Intracellular Trafficking and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.
Kim
Soo Jin
SJ
Lee
Yong Jik
YJ
Jin
Jing Bo
JB
Hwang
Inhwan
I
D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins; D002462:Cell Membrane / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006720:Homozygote; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D010641:Phenotype; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D021381:Protein Transport
D029681:Arabidopsis Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; C403444:TOC159 protein, Arabidopsis; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases