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An ankyrin repeat domain of AKR2 drives chloroplast targeting through coincident binding of two chloroplast lipids.

ABSTRACT: In organellogenesis of the chloroplast from endosymbiotic cyanobacteria, the establishment of protein-targeting mechanisms to the chloroplast should have been pivotal. However, it is still mysterious how these mechanisms were established and how they work in plant cells. Here we show that AKR2A, the cytosolic targeting factor for chloroplast outer membrane (COM) proteins, evolved from the ankyrin repeat domain (ARD) of the host cell by stepwise extensions of its N-terminal domain and that two lipids, monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG), of the endosymbiont were selected to function as the AKR2A receptor. Structural analysis, molecular modeling, and mutational analysis of the ARD identified two adjacent sites for coincidental and synergistic binding of MGDG and PG. Based on these findings, we propose that the targeting mechanism of COM proteins was established using components from both the endosymbiont and host cell through a modification of the protein-protein-interacting ARD into a lipid binding domain.

SUBMITTER: Kim DH

PROVIDER: S-EPMC4170656 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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An ankyrin repeat domain of AKR2 drives chloroplast targeting through coincident binding of two chloroplast lipids.

Kim Dae Heon DH Park Mi-Jeong MJ Gwon Gwang Hyeon GH Silkov Antonina A Xu Zheng-Yi ZY Yang Eun Chan EC Song Seohyeon S Song Kyungyoung K Kim Younghyun Y Yoon Hwan Su HS Honig Barry B Cho Wonhwa W Cho Yunje Y Hwang Inhwan I

Developmental cell 20140901 5

In organellogenesis of the chloroplast from endosymbiotic cyanobacteria, the establishment of protein-targeting mechanisms to the chloroplast should have been pivotal. However, it is still mysterious how these mechanisms were established and how they work in plant cells. Here we show that AKR2A, the cytosolic targeting factor for chloroplast outer membrane (COM) proteins, evolved from the ankyrin repeat domain (ARD) of the host cell by stepwise extensions of its N-terminal domain and that two li ...[more]

PMID: 25203210

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Project description:Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of I?B?, the temporally regulated inhibitor of canonical NF?B signaling. Under native conditions, most of the I?B? molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NF?B(RelA/p50)-I?B? complex. Similar high FRET efficiencies were found when the I?B? molecules were either free or in complex with NF?B(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound I?B? molecules (0.67) than for the free I?B? molecules (0.74), apparently indicating that binding of NF?B(RelA/p50) stretches the ARD of I?B?. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NF?B(RelA/p50) and I?B? encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on I?B? as well as I?B? bound to dimerization-domain-only constructs or full-length NF?B(RelA/p50). Although we expected the stretched I?B? to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NF?B NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the I?B? ARD induced by interactions of the I?B? proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

Dataset Information

An ankyrin repeat domain of AKR2 drives chloroplast targeting through coincident binding of two chloroplast lipids.

Publications

An ankyrin repeat domain of AKR2 drives chloroplast targeting through coincident binding of two chloroplast lipids.

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