The Atg1 complex, comprising Atg1, Atg13, Atg17, Atg29, and Atg31, is a key initiator of autophagy. The Atg17-Atg31-Atg29 subcomplex is constitutively present at the phagophore assembly site (PAS), while Atg1 and Atg13 join the complex when autophagy is triggered by starvation or other signals. We sought to understand the energetics and dynamics of assembly using isothermal titration calorimetry (ITC), sedimentation velocity analytical ultracentrifugation, and hydrogen-deuterium exchange (HDX). We showed that the membrane and Atg13-binding domain of Atg1, Atg1EAT, is dynamic on its own, but is rigidified in its high-affinity (∼100 nM) complex with Atg13. Atg1EAT and Atg13 form a 2:2 dimeric assembly and together associate with lower affinity (∼10 μM) with the 2:2:2 Atg17-Atg31-Atg29 complex. These results lead to an overall model for the assembly pathway of the Atg1 complex. The model highlights the Atg13-Atg17 binding event as the weakest link in the assembly process and thus as a natural regulatory checkpoint.

Yeast macroautophagy begins with the de novo formation of a double-membrane phagophore at the preautophagosomal structure/phagophore assembly site (PAS), followed by its expansion into the autophagosome responsible for cargo engulfment. The kinase Atg1 is recruited to the PAS by Atg13 through interactions between the EAT domain of the former and the tMIM motif of the latter. Mass-spectrometry data have shown that, in the absence of Atg13, the EAT domain structure is strikingly dynamic, but the function of this Atg13-free dynamic state has been unclear. We used structure-based mutational analysis and quantitative and superresolution microscopy to show that Atg1 is present on autophagic puncta at, on average, twice the stoichiometry of Atg13. Moreover, Atg1 colocalizes with the expanding autophagosome in a manner dependent on Atg8 but not Atg13. We used isothermal titration calorimetry and crystal structure information to design an EAT domain mutant allele ATG1^DD that selectively perturbs the function of the Atg13-free state. Atg1^DD shows reduced PAS formation and does not support phagophore expansion, showing that the EAT domain has an essential function that is separate from its Atg13-dependent role in autophagy initiation.

The initial stage of CRISPR–Cas immunity involves the acquisition of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved amongst all CRISPR–Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å resolution crystal structure of the Cas1–Cas2 complex. Mutations that perturb Cas1–Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Unlike Cas1, active site mutants of Cas2 can still acquire new spacers indicating a non-enzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1–Cas2 complexes specify sites of CRISPR spacer integration.

The autophagy-related 1 (Atg1) complex of Saccharomyces cerevisiae has a central role in the initiation of autophagy following starvation and TORC1 inactivation. The complex consists of the protein kinase Atg1, the TORC1 substrate Atg13, and the trimeric Atg17-Atg31-Atg29 scaffolding subcomplex. Autophagy is triggered when Atg1 and Atg13 assemble with the trimeric scaffold. Here we show by hydrogen-deuterium exchange coupled to mass spectrometry that the mutually interacting Atg1 early autophagy targeting/tethering domain and the Atg13 central domain are highly dynamic in isolation but together form a stable complex with ∼ 100-nM affinity. The Atg1-Atg13 complex in turn binds as a unit to the Atg17-Atg31-Atg29 scaffold with ∼ 10-μM affinity via Atg13. The resulting complex consists primarily of a dimer of pentamers in solution. These results lead to a model for autophagy initiation in which Atg1 and Atg13 are tightly associated with one another and assemble transiently into the pentameric Atg1 complex during starvation.