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. 2020 Feb 7;23(3):100889. doi: 10.1016/j.isci.2020.100889

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© 2020 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Specific Positively Charged Lysine-Arginine Pairs in the N-terminal Region of AtTSPO Mediate Interaction with PIP2;7 In Vivo

(A) Representative confocal images of tobacco epidermal cells transiently coexpressing Venus_N-PIP2;7 and Venus_C-AtTSPO or Venus_N-PIP2;7 and Venus_C-AtTSPO point mutants. Xylosyltransferase-mTurquoise fluorescent chimera (magenta) served as a cell transfection control (Golgi marker) and for signal quantification. Full-length PIP2;7 and AtTSPO served as a positive control for the BiFC signal. Low-magnification images qualitatively demonstrate the occurrence and distribution of BiFC (green) in the transfected area, and insets of high-magnification images show Golgi stacks (arrowheads). For PIP2;7-AtTSPO^K35A/R36A, colocalization of the BiFC signal and Golgi marker was not detected. Bars = 20 μm and 5 μm for low and high magnification, respectively. Experiments were repeated three times.

(B) Signal quantification showing a drastic reduction in BIFC between PIP2;7 and AtTSPO mutants compared with full-length AtTSPO. Statistical analysis was based on one-way ANOVA followed by Tukey's tests (***p < 0.001). Bars represent means +/− SD. See transparent methods section for quantification procedure.

(C) Hypothetical model of PI(4,5)P₂-dependent PIP2;7 downregulation by AtTSPO during stress. Under normal conditions (left), synthesized PiP2;7 is targeted to the PM through the biosynthetic secretory pathway. Under osmotic stress conditions (right), expressed AtTSPO is targeted to the ER and Golgi membranes.AtTSPO stimulates PLC activity, depleting PI(4,5)P₂ at the PM and generating DAG and PA that modulate the activity of PP2C phosphatases involved in ABA-dependent reduction of water loss through stomata. Decreased PI(4,5)P₂ at the PM prevents recruitment of PIP2;7-containing vesicles. Golgi/ER-localized AtTSPO binds PI(4,5)P₂ synthesized de novo in these compartments, inducing structural changes needed for recognition and interaction with PIP2;7 en route to the PM, causing redirection to nascent autophagosomes. AtTSPO cannot directly deplete PI(4,5)P₂ from the PM but may form a protein complex that recruits PIP5K to organelle/autophagosome initiation sites. AtTSPO-mediated enrichment of ER/Golgi membranes with PI(4,5)P₂ may initiate autophagy. Autophagosomal membranes also recruit PIP5K that generates PI(4,5)P₂ and the autophagy regulator Atg14/Barkor that interacts with both the enzyme and PI(4,5)P₂. PI(4,5)P₂ binding to Atg14 regulates its interaction with the Vps34 complex catalyzing PI3P synthesis at autophagosome initiation sites. The presence of the AtTSPO-PI(4,5)P₂-PIP2;7 complex in ER/Golgi membranes and the phagophore containing PI(4,5)P₂ and Atg8 may be close enough to allow lipid-protein and protein-protein interactions.