Biomolecular condensates of ATG18 reshape ER to promote autophagy in plants

Description

Autophagosomes originate from and maintain association with the endoplasmic reticulum (ER) during their formation, yet how these processes are molecularly coordinated in plants remains poorly understood. Here, we demonstrate that Arabidopsis ATG18a, a key organizer of early autophagosome formation, undergoes phase separation to form biomolecular condensates on the ER membrane, which progress from highly mobile droplets to stable ring-like structures, while the ER is reshaped. We discovered that ATG18a condensates work together with RHD3, an ER membrane-shaping protein, with RABC1 serving as a molecular linker between them. Importantly, RABC1 facilitates both RHD3 assembly necessary for formation of ringlike ER structures and its interaction with ATG18a condensates. These findings reveal a novel mechanism whereby biomolecular condensates, work together with membrane shaping proteins to reshape specialized membrane domains through wetting interactions, providing fundamental insights into autophagosome formation in plant stress responses. Here all original and full western blots are included.

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636 Co-immunoprecipitation 637 Leaves of N. benthamiana infiltrated with various protein combinations, or Arabidopsis seedlings 638 subjected to salt stress treatment were ground in liquid nitrogen. Proteins were then extracted using a 639 buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) IGEPAL CA- 640 630, and 1% (v/v) Protease Inhibitor Cocktail. The extracts were centrifuged at 17,000 g for 10 minutes 641 at 4°C. The supernatant was collected and incubated with 25 mL Anti-GFP or Anti-mCherry Nanobody 642 Magarose Beads (AlpaLifeBio) for 1 hour at 4°C. The beads were magnetically separated and washed 643 thrice with wash buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl). The beads were then resuspended in 644 100 μL 2× SDS-loading buffer and boiled for 10 minutes for subsequent Western blot analysis. 645 646 Western blot 647 Protein samples were subjected to electrophoresis on a 10% SDS-PAGE gel and subsequently transferred 648 onto a PVDF membrane. The membrane was probed with the following primary antibodies at a dilution 649 of 1:5000: mouse anti-GFP (AE012, Abclonal), mouse anti-mCherry (EA012, ELK Biotechnology), 650 mouse anti-HA (66006-2-Ig, Proteintech), rabbit anti-RHD3 (PHY0765S, Phytoab), rabbit anti-NBR1 651 (PHY3164S, Phytoab), rabbit anti-BiP2 (PHY1481A, Phytoab), rabbit anti-PEX12 (PHY7126S, 652 Phytoab), and rabbit anti-CYC1 (PHY0566A, Phytoab). Goat anti-mouse IgG (SA002, ELK 653 Biotechnology, 1:10000) and goat anti-rabbit IgG (AS09 602, Agrisera, 1:10000) were used as secondary 654 antibodies. The signals were visualized using Invitrogen Novex ECL (HRP Chemiluminescent Substrate 655 Reagent Kit) according to the manufacturer's instructions.

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