Journal: Molecular Cell

Article Title: Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation

doi: 10.1016/j.molcel.2021.10.024

Figure Lengend Snippet: Atg8-PE stimulates Atg1 kinase activity (A) Atg1-mediated phosphorylation of catalytically inactive Atg1 D211A was monitored in the presence of either 4 μM lipidated Atg8 ΔR117 (Atg8 ΔR117 -PE) or non-lipidated Atg8 ΔG116/ΔR117 (upper panel). Before the addition of Atg1, Atg8 ΔR117 and Atg8 ΔG116/ΔR117 were incubated with Atg7, Atg3, and liposomes (55% PE, 35% phosphatidylcholine [PC], and 10% phosphatidylinositol [PI]) to promote Atg8 ΔR117 lipidation. Atg1 D211A (2.5 μM) was added, and in vitro kinase assays were started by the addition of WT Atg1 (50 nM). Control reactions (lower panel) did not contain Atg1 D211A reporting only on Atg1 autophosphorylation. Time points were analyzed by SDS-PAGE and autoradiography. (B) Atg1-dependent phosphorylation of Atg8 and Atg3 was monitored in a time-dependent manner in the presence of either Atg8 (Atg8 ΔR117 ) or Atg8-PE (Atg8 ΔR117 -PE). Atg8 was lipidated before starting the in vitro kinase assays by addition of pre-phosphorylated Atg1. Control reactions lacked either Atg7 or Atg8. Time points were analyzed by SDS-PAGE and autoradiography. Asterisk, Atg8-Atg3 conjugate. (C) Relative quantification of Atg1-dependent Atg3 and Atg8/Atg8-PE phosphorylation rates (n = 3) in the presence of Atg8 (by omitting either Atg7 or liposomes) or Atg8-PE. Atg3 phosphorylation rates were also quantified in the absence of Atg8. Representative autoradiographs used for quantification are shown in Figure S2 A. (D) Relative rate increase of Atg1-dependent substrate phosphorylation in the presence of Atg8-PE. Atg8-PE-dependent phosphorylation rates were compared to control reactions devoid of Atg7 or liposomes. Relative phosphorylation rates for the indicated Atg proteins were determined using Atg8, Atg3, Atg12–Atg5-Atg16, Vps34 Atg14/Atg38 , and Atg4 C147A as substrates. Atg1 was phosphorylated before substrate addition. (E) Schematic representation of S. cerevisiae Atg1 highlighting the Atg8-interacting motif (AIM) (salmon), kinase, and MIT domains (purple and green, respectively). Atg1 autophosphorylation sites and corresponding phosphomimicking mutations are shown in red. Atg1-dependent phosphorylation of Atg3 was assayed in the presence of WT (AIM WT ) or mutated AIM peptides (AIM AA and AIM WT/S > D ). Competition experiments were carried out in the presence of either Atg8 (bottom row) or Atg8-PE (top row). Longer time points were used to monitor Atg3 phosphorylation in the presence of Atg8. (F) Quantification of the relative Atg1-dependent Atg3 phosphorylation rates in arbitrary units (a.u.) in the presence of the indicated AIM peptides and either Atg8 or Atg8-PE. (G) Phosphorylation of Atg3 and Atg8/Atg8-PE was compared between WT Atg1 and Atg1 ΔAIM/S > A (Atg1 S418A/S421A/S424A/Y429A/V432A ) in the presence of either Atg8 or Atg8-PE. Different time points are shown for Atg8 and Atg8-PE-containing reactions. (H) Quantification of the relative Atg1 and Atg1 ΔAIM/S > A -dependent phosphorylation rates for Atg3 and Atg8/Atg8-PE in the presence of either Atg8 or Atg8-PE (n = 3). (I) Localization of neonGreen-tagged Atg1 and Atg1 ΔAIM/S > A in nitrogen-starved atg11Δ cells overexpressing tagBFP-Ape1. A single z stack is shown. Scale bar, 2 μm. (J) Atg1, Atg1 ΔAIM (Atg1 Y429A/V432A ), and Atg1 ΔAIM/S > A pull-down experiments using SF-tagged Atg8 as bait (I Atg8 ) were analyzed by SDS-PAGE. Atg1 preparations were either autophosphorylated (ATP/Mg 2+ ) or dephosphorylated using λ-PP (−). To avoid differential Atg8 phosphorylation, all Atg1 preparations were treated with apyrase before addition to Atg8-coated FLAG resin. Ubiquitin was used as a specificity control.

Article Snippet: The following lipids were purchased from Avanti Polar Lipids: bovine L-α-phosphatidylethanolamine (PE; 840026C), bovine L-α-phosphatidylcholine (PC; 840055C), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; 850375), bovine L-α-phosphatidylinositol (PI; 840042P) and brain L-α-phosphatidylserine (PS; 840032C).

Techniques: Activity Assay, Incubation, In Vitro, SDS Page, Autoradiography, Relative Rate

Journal: Molecular Cell

Article Title: Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation

doi: 10.1016/j.molcel.2021.10.024

Figure Lengend Snippet:

Article Snippet: The following lipids were purchased from Avanti Polar Lipids: bovine L-α-phosphatidylethanolamine (PE; 840026C), bovine L-α-phosphatidylcholine (PC; 840055C), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; 850375), bovine L-α-phosphatidylinositol (PI; 840042P) and brain L-α-phosphatidylserine (PS; 840032C).

Techniques: Subcloning, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, PCR Cloning, Plasmid Preparation, Gel Extraction, Mass Spectrometry, Software, Inverted Microscopy, Protein Purification, Staining, Western Blot

Journal: Molecular Cell

Article Title: Human Condensin I and II Drive Extensive ATP-Dependent Compaction of Nucleosome-Bound DNA

doi: 10.1016/j.molcel.2020.04.026

Figure Lengend Snippet:

Article Snippet: 18:1 PEG2000 PE , Avanti Polar Lipids , 880130P-25mg.

Techniques: Virus, Recombinant, Protease Inhibitor, Microscopy, Lambda DNA Preparation, Mass Spectrometry, Sequencing, Labeling, Strep-tag, Software, Inverted Microscopy