Abstract
Toc75 plays a central role in chloroplast biogenesis in plants as the membrane channel of the protein import translocon at the outer envelope of chloroplasts (TOC). Toc75 is a member of the Omp85 family of bacterial and organellar membrane insertases, characterized by N-terminal POTRA (polypeptide-transport associated) domains and C-terminal membrane-integrated β-barrels. We demonstrate that the Toc75 POTRA domains are essential for protein import and contribute to interactions with TOC receptors, thereby coupling preprotein recognition at the chloroplast surface with membrane translocation. The POTRA domains also interact with preproteins and mediate the recruitment of molecular chaperones in the intermembrane space to facilitate membrane transport. Our studies are consistent with the multi-functional roles of POTRA domains observed in other Omp85 family members and demonstrate that the domains of Toc75 have evolved unique properties specific to the acquisition of protein import during endosymbiotic evolution of the TOC system in plastids.
Highlights
The biogenesis and function of plastids are reliant on the coordinate expression and selective import of >2500 different nucleus-encoded proteins (Ling and Jarvis, 2015, Shi and Theg, 2013)
To investigate the role of POTRA domains in the function of Toc75, we introduced a series of inframe internal deletion mutations in a TOC75-III genomic construct to encode proteins lacking one (Toc75DP1), two (Toc75DP1-2) or all three (Toc75DP1-3) predicted POTRA domains (Figure 1A), and tested their ability to complement the lethal phenotype of the toc75-III-1 null mutant
Transformants were selected for hygromycin resistance linked to the toc75-III-1 T-DNA insertion and for the DsRed fluorescence marker linked to the TOC75 and POTRA-deletion constructs
Summary
The biogenesis and function of plastids are reliant on the coordinate expression and selective import of >2500 different nucleus-encoded proteins (Ling and Jarvis, 2015, Shi and Theg, 2013). TOC and TIC complexes contain selective protein conducting channels, which align via physical association between the complexes to provide simultaneous membrane translocation of preproteins from the cytoplasm to the plastid interior (Paila et al, 2015, Shi and Theg, 2013, Ling and Jarvis, 2015). The evolution of the TOC and TIC system during endosymbiosis required the adaptation or acquisition of protein conducting channels, receptors and molecular chaperones at both envelope membranes to provide a unidirectional conduit for the transport of nuclear encoded polypeptides into the organelle. Toc assembles with two integral membrane receptor GTPases, Toc159 and Toc, which recognize plastid preproteins in the cytoplasm and control access of
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