p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.

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Tryptophan-derived, indolic metabolites possess diverse functions in Arabidopsis innate immunity to microbial pathogen infection. Here, we investigate the functional role and regulatory characteristics of indolic metabolism in Arabidopsis systemic acquired resistance (SAR) triggered by the bacterial pathogen Pseudomonas syringae. Indolic metabolism is broadly activated in both P. syringae-inoculated and distant, non-inoculated leaves. At inoculation sites, camalexin, indol-3-ylmethylamine (I3A), and indole-3-carboxylic acid (ICA) are the major accumulating compounds. Camalexin accumulation is positively affected by MYB122, and the cytochrome P450 genes CYP81F1 and CYP81F2. Local I3A production, by contrast, occurs via indole glucosinolate breakdown by PEN2- dependent and independent pathways. Moreover, exogenous application of the defense hormone salicylic acid stimulates I3A generation at the expense of its precursor indol-3-ylmethylglucosinolate (I3M), and the SAR regulator pipecolic acid primes plants for enhanced P. syringae-induced activation of distinct branches of indolic metabolism. In uninfected systemic tissue, the metabolic response is more specific and associated with enhanced levels of the indolics I3A, ICA, and indole-3-carbaldehyde (ICC). Systemic indole accumulation fully depends on functional CYP79B2/3, PEN2, and MYB34/51/122, and requires functional SAR signaling. Genetic analyses suggest that systemically elevated indoles are dispensable for SAR and associated systemic increases of salicylic acid. However, soil-grown but not hydroponically -cultivated cyp79b2/3 and pen2 plants, both defective in indolic secondary metabolism, exhibit pre-induced immunity, which abrogates their intrinsic ability to induce SAR.

ESCRT-III proteins mediate a range of cellular membrane remodeling activities such as multivesicular body biogenesis, cytokinesis, and viral release. Critical to these processes is the assembly of ESCRT-III subunits into polymeric structures. In this study, we determined the cryo-EM structure of a helical assembly of Saccharomyces cerevisiae Vps24 at 3.2-Å resolution and found that Vps24 adopts an elongated open conformation. Vps24 forms a domain-swapped dimer extended into protofilaments that associate into a double-stranded apolar filament. We demonstrate that, upon binding negatively charged lipids, Vps24 homopolymer filaments undergo partial disassembly into shorter filament fragments and oligomers. Upon the addition of Vps24, Vps2, and Snf7, liposomes are deformed into neck and tubular structures by an ESCRT-III heteropolymer coat. The filamentous Vps24 homopolymer assembly structure and interaction studies reveal how Vps24 could introduce unique geometric properties to mixed-type ESCRT-III heteropolymers and contribute to the process of membrane scission events.