Tobias Bläske

Abstract Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca 2+ -dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.

The abundance of plasma membrane-resident receptors and transporters has to be tightly regulated by ubiquitin-mediated endosomal degradation for the proper coordination of environmental stimuli and intracellular signaling. Arabidopsis OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 are plasma membrane-localized deubiquitylating enzymes (DUBs) that bind to phospholipids through a polybasic motif in the OTU domain. Here we show that the DUB activity of OTU11 and OTU12 towards K63-linked ubiquitin is stimulated by binding to lipid membranes containing anionic lipids. In addition, we show that the DUB activity of OTU11 against K6- and K11-linkages is also stimulated by anionic lipids, and that OTU11 and OTU12 can modulate the endosomal degradation of a model cargo and the auxin efflux transporter PIN2-GFP in vivo. Our results suggest that the catalytic activity of OTU11 and OTU12 is tightly connected to their ability to bind membranes and that OTU11 and OTU12 are involved in the fine-tuning of plasma membrane proteins in Arabidopsis.

Clathrin coated vesicles (CCVs) mediate endocytosis of plasma membrane proteins and deliver their content to the endosomes for either subsequent recycling to the plasma membrane or transport to the vacuole for degradation. CCVs assemble also at the trans-Golgi network (TGN) and is responsible for the transport of proteins to other membranes. Oligomerization of clathrin and recruitment of adaptor protein complexes promote the budding and the release of CCVs, however, many of the details during plant CCV formation are not completely elucidated. The analysis of isolated CCVs is therefore important to better understand the formation of plant CCVs, their cargos and the regulation of clathrin-mediated transport processes. In this article, we describe an optimized method to isolate CCVs from Arabidopsis thaliana seedlings.

Abstract Deubiquitinases (DUBs) remove ubiquitin modifications from proteins in a substrate- or linkage-selective manner and regulate numerous cell-biological processes, including endocytosis. By performing homology-agnostic bioinformatical screens for undescribed deubiquitinase classes, we identified the plant-specific DUC ( d e u biquitinase with C 2) family, whose members are highly selective for cleaving K63-linked ubiquitin chains. The crystal structure of the Arabidopsis member AtDUC1 reveals that DUC enzymes display a papain-like fold with a characteristic DUB-like active site, despite the apparent absence of sequence similarity to other deubiquitinase families. The K63 linkage specificity is attributed to the recognition of both distal and proximal ubiquitin units via highly conserved contact residues. Arabidopsis DUC members localize to the plasma membrane by virtue of their lipid-binding C2-like domains. In protoplast experiments, all three Arabidopsis DUC enzymes demonstrate the capacity to stabilize an endocytotic model cargo at the plasma membrane, a process that requires both the C2-mediated membrane association and catalytic DUB activity.

Abstract Autophagy is a major eukaryotic degradation and recycling pathway, which eliminates damaged cellular components and provides energy and building blocks especially under adverse environmental conditions. While the core autophagy machinery has been extensively studied, autophagy initiation and regulation remain less understood. In this study, we show that genes encoding the core autophagy initiation complex, ATG1 , ATG11 , ATG13 , and ATG101 , are transcriptionally regulated by sugars. To analyze the expression of these genes, we took advantage of Arabidopsis thaliana accessions, which display strong variations in their responses to darkness-induced fixed carbon deprivation. One of the components of the initiation complex, ATG101 , has multiple sugar-related elements, and was repressed by sugar. Moreover, ATG101 was induced stronger upon starvation in carbon starvation-resistant accessions when compared to carbon starvation-sensitive accessions. We identified three single nucleotide polymorphisms in the genomic region of ATG101 which are associated with the carbon-starvation phenotype. Further analyses through complementation of the atg101 mutant with different variants of ATG101 demonstrated that the single nucleotide polymorphism alleles contribute differently to ATG101 expression regulation, response to sugar, and phenotype recovery. Taken together, our data suggest that modulation of ATG101 by sugar is part of a multi-layered mechanism regulating the sensitivity to carbon starvation in Arabidopsis.