Structural basis of starvation-induced assembly of the autophagy initiation complex

Abstract

Assembly of the preautophagosomal structure (PAS) is essential for autophagy initiation in yeast. Starvation-induced dephosphorylation of Atg13 is required for the formation of the Atg1-Atg13-Atg17-Atg29-Atg31 complex (Atg1 complex), a prerequisite for PAS assembly. However, molecular details underlying these events have not been established. Here we studied the interactions of yeast Atg13 with Atg1 and Atg17 by X-ray crystallography. Atg13 binds tandem microtubule interacting and transport domains in Atg1, using an elongated helix-loop-helix region. Atg13 also binds Atg17, using a short region, thereby bridging Atg1 and Atg17 and leading to Atg1-complex formation. Dephosphorylation of specific serines in Atg13 enhanced its interaction with not only Atg1 but also Atg17. These observations update the autophagy-initiation model as follows: upon starvation, dephosphorylated Atg13 binds both Atg1 and Atg17, and this promotes PAS assembly and autophagy progression.

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

“…16,36 More recently, an Atg17 binding region (17BR) of Atg13 was further mapped to residues 424 to 436 through a combination of crystallographic and biochemical methods. 23 Our CXMS data showed that the Atg13 [CTD] directly crosslinks to Atg17 in the context of the pentameric assembly. The Atg17-Atg13[CTD] interactions represented the most abundant intersubunit peptide group from our dataset ( Table S1).…”

“…Furthermore, recent phosphoproteomics analysis of S. cerevisiae Atg13 showed that these 3 serine residues are phosphorylated under nonstarvation conditions. 23 To determine the effects of phosphorylation on the interaction between Atg13 17BR2 and Atg17, we generated nonphosphorylatable ([3SA]) and phosphomimetic ([3SD]) mutations on Atg13 (residues 630 to 694) and analyzed these constructs by coprecipitation experiments. We found that while phosphomimetic mutation (3SD) did not affect the ability of Atg13 17BR2 to bind Atg17, the nonphosphorylatable mutation (3SA) significantly enhanced this interaction (Fig.…”

“…24,25 Nutrient starvation leads to rapid Atg13 dephosphorylation, an event that has been proposed to increase the binding affinity of Atg13 to Atg1 and thereby promote the formation of the full Atg1 complex. 23,24 A separate study, however, suggests that the Atg1 complex is preformed with its structural integrity unaffected by the phosphorylation state of Atg13. 26 The majority of Atg13's phosphorylation sites map to the C-terminal domain (residues 260 to 738), a region that is predicted to be intrinsically unstructured.…”

“…22 More recently, the C-terminal domain of K. marxianus Atg1 (residues 587 to 897) was co-crystallized in complex with a central fragment of Atg13 (residues 460 to 521). 23 Although these studies have provided new insights into the protein-protein interactions mediating complex formation, many questions regarding complex assembly still remain. Notably, the roles of the intrinsically unstructured domains of Atg29, Atg31, and Atg13 in complex formation and regulation have not been investigated as these domains are highly challenging to be studied by X-ray crystallography.…”

“…21,22 However, recent crystallographic analysis of the Kluyveromyces marxianus Atg1 C-terminal domain showed that its structure does not resemble canonical membrane-curvature sensors. 23 Atg13 functions as a major positive regulator for the Atg1 protein kinase. Atg13 is highly phosphorylated during nutrient-rich conditions by protein kinase A and the target of rapamycin (TOR) complex 1 (TORC1).…”