DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis.Here we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, hence redefining the architecture of the active eukaryotic replisome..

Summary Macroautophagy (hereafter autophagy) is an evolutionarily conserved process in which portions of the cytoplasm are engulfed, degraded and subsequently recycled. The Atg17-Atg31-Atg29 complex translocates to the phagophore assembly site (PAS), where an autophagosome forms, at a very early stage of autophagy, playing a vital role in autophagy induction. Here, we identified a novel role of this complex in a late stage of autophagy where it coordinates with Atg11 to regulate autophagy-specific fusion with the vacuole. Atg17 and Atg11 interact with the vacuolar SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) Vam7 independent of each other. Several hydrophobic residues in helix 1 and helix 4 of Atg17 and the SNARE domain of Vam7 mediate the Atg17-Vam7 interaction. An F317D mutation of Atg17, which diminished its interaction with Vam7 without affecting its interaction with Atg13 or Atg31, leads to a defect in the fusion of autophagosomes with the vacuole and decreased autophagy activity. These results provide the first demonstration that the Atg17-Atg31-Atg29 complex functions in both early and late stages of autophagy, and provides a mechanistic explanation for the coordination of autophagosome completion and fusion with the vacuole.

(2015) Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms, Autophagy, 11:6, 891-905, DOI: 10.1080/15548627.2015 The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1 [CTD]) and Atg13 (Atg13 [CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1 [CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, hence redefining the architecture of the active eukaryotic replisome.