Abbreviations: ATG, autophagy-related; Bafi, bafilomycin A 1 ; BSA, bovine serum albumin; C. elegans, Caenorhabditis elegans; CAL-COCO2, calcium binding and coiled-coil domain 2; DAPI, 4', 6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; DPH, 1, 6-diphenyl-1, 3, 5-hexatriene; eV, empty vector; FEZ, fasciculation and elongation protein zeta; GABARAP, GABA(A) receptor-associated protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; MAP1LC3, microtubule-associated protein 1 light chain 3; NBR1, neighbor of BRCA1 gene 1; PE, phosphatidylethanolamine; PBS, phosphate-buffered saline; RABGAP, RAB GTPase activating protein; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TBC domain, TRE2-BUB2-CDC16 domain.Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.

Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens. RNA viruses typically have high mutation rates, resulting in frequent production of protein variants with compromised biophysical properties. Their evolution is necessarily constrained by the consequent challenge to protein folding and function. We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants, serving as a critical force shaping viral evolution. Here, we test that hypothesis by propagating influenza in host cells displaying chemically-controlled, divergent proteostasis environments. We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis. These findings provide new insights into features of host-pathogen interactions that shape viral evolution, and into the potential design of host proteostasis-targeted antiviral therapeutics that are refractory to resistance.

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Autophagy is a lysosomal degradation pathway that mediates protein and organelle turnover and maintains cellular homeostasis. Autophagosomes transport cargo to lysosomes and their formation is dependent on an appropriate lipid supply. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet (LD) metabolism, resulting in an impaired fatty acid mobilization. The reduced LD-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network, which include increased ATG2B expression and ATG12-ATG5 conjugate formation as well as enhanced ATG2B and ATG9A phosphorylation. Phosphorylation of ATG9A directs this transmembrane protein to the site of autophagosome formation and this particular modification is sufficient to rescue autophagic activity under basal conditions in the absence of RAB18. However, it is incapable of enabling an increased autophagy under inductive conditions. Thus, we illustrate the role of RAB18 in connecting LDs and autophagy, further emphasize the importance of LD-derived lipids for the degradative pathway, and characterize an ATG9A phosphorylation-dependent autophagy rescue mechanism as an adaptive response that maintains autophagy under conditions of reduced LD-derived lipid availability. Keywords ATG9A phosphorylation / autophagosome formation / autophagy / lipid droplets / RAB18Introduction Macroautophagy (hereafter referred to as autophagy) is a eukaryotic lysosomal degradation pathway that mediates protein and organelle turnover and maintains cellular homeostasis [1].Autophagic cargo is transported in vesicles, so-called autophagosomes, to lysosomes for degradation and recycling of building blocks. The generation of autophagosomes is dynamic and increases within minutes upon autophagy induction. Several conditions, including nutrient deprivation or rapamycin treatment, stimulate autophagy and result in activation of the AMP-activated kinase PRKAA1 and/or inhibition of the kinase MTOR [2]. Subsequently, ULK1 kinase is activated by phosphorylation, which is a pre-requisite for the generation of autophagosomes [3]. The synthesis of double-membraned autophagosomes starts with a precursor membrane, the phagophore, which originates from specific omega-shaped domains (omegasomes) in the ER membrane [4-6]. The phagophore elongates by the addition of lipids or membranes until it finally closes to form an autophagosome, which completely incorporates the autophagic cargo. This process involves a cascade of proteins, including two ubiquitin-like conjugations systems, that mediate the formation of the ATG12-ATG5/ATG16L1 protein complex as well as the lipidation of Atg8 family members, such as MAP1LC3B (shortly LC3) [7]. Indeed, the ATG12-ATG5/ATG16L1 complex localizes to the phagophore membrane and shows E3 ligase-like activity that promotes the final step of Atg8 lipidation [8-10]. Lipid-conjugated Atg8 proteins associate with the growing phagophore membrane and are essential for autophagosome formation [11]. The proteins stay (partial...