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1 Architecture and Dynamics of the Autophagic Phosphatidylinositol 3 1 Architecture and Dynamics of the Autophagic Phosphatidylinositol 3-Kinase 2 Complex 3 4 Sulochanadevi Baskaran1,*, Lars-Anders Carlson1,*, Goran Stjepanovic1,*, Lindsey N. 5 Young1, Do Jin Kim1, Patricia Grob1,2, Robin E. Stanley4,5, Eva Nogales1,2,3, and James H. 6 Hurley1,3 7 8 1Department of Molecular and Cell Biology and California Institute for Quantitative 9 Biosciences, University of California, Berkeley, Berkeley, CA 94720 10 11 2Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 12 94720, USA 13 14 3Life Sciences Division, Lawrence Berkeley National Lab, Berkeley, CA 94720, USA 15 16 17 4Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney 18 Diseases, National Institutes of Health, Bethesda, MD 20892, USA 19 20 5Present address: Laboratory of Signal Transduction, National Institute of Environmental 21 Health Sciences, Department of Health and Human Services, National Institutes of 22 Health, Research Triangle Park, NC 27709, USA 23 24 Running title: EM Structure of the Autophagic PI3KC3 25 26 *equal contribution 27 28 correspondence: [email protected] or [email protected] 29 1 30 Abstract 31 The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in 32 early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the 33 tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of 34 the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V- 35 shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were 36 mapped by MBP tagging. The dynamics of the complex were defined using hydrogen- 37 deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the 38 VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 39 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase 40 domain is ejected from the complex and VPS15 pivots at the base of the V. The N- 41 terminus of BECN1, the target for signaling inputs that regulate PI3KC3 activity, resides 42 near the pivot point. These observations provide a framework for understanding the 43 allosteric regulation of lipid kinase activity. 44 45 2 46 Introduction 47 Macroautophagy (hereafter, “autophagy”) is a conserved eukaryotic pathway for cellular 48 self-preservation through cellular self-consumption (Green and Levine, 2014; Mizushima 49 et al., 2011; Reggiori and Klionsky, 2013; Rubinsztein et al., 2012b). The most ancient 50 role for autophagy is in survival during starvation. Bulk cytosol is captured in a growing 51 double membrane structure termed the phagophore. Upon sealing, the resulting double 52 membrane vesicle is known as an autophagosome. The autophagosome fuses with the 53 lysosome or vacuole, resulting in the degradation of its contents and the recycling of 54 biosynthetic precursors by export across the lysosomal membrane. In higher eukaryotes, 55 autophagy has acquired many additional roles in cellular protection. For example, the 56 core autophagy protein BECN1 (Beclin 1) is a tumor suppressor. BECN1 is deleted in 40- 57 75% of human breast, ovarian, and prostate cancers (Liang et al., 1999). Autophagy is 58 also thought, under most conditions, to protect cells from the accumulation of pathogenic 59 inclusions that can lead to Huntington’s, Parkinson’s, and other neurodegenerative 60 diseases (Nixon, 2013; Rubinsztein et al., 2012a). 61 During the initiation of autophagy, an autophagy-specific form of the class III 62 phosphatidylinositol 3-kinase complex (PI3KC3-C1) is activated and recruited to the site 63 of phagophore nucleation. Class III PI3Ks synthesize phosphatidylinositol 3-phosphate 64 (PI(3)P) from phosphatidylinositol (PI), as compared to the class I and II PI3Ks that 65 generate PI(3,4,5)P3 and PI(3,4)P2, respectively (Backer, 2008). In yeast, where the 66 complex was first characterized, there are two PI 3-kinase complexes, known as 67 complexes I and II, with clear-cut distinctions in their functions (Kihara et al., 2001). 68 Both yeast complexes contain the core subunits Vps34, Vps15, and Atg6. Vps34 contains 69 the catalytic domain responsible for lipid kinase activity, as well as a putative lipid 70 binding C2 domain and a helical domain (Miller et al., 2010; Schu et al., 1993). Vps15 is 71 a large (150 kDa) protein essential for the activity of the complex (Stack et al., 1993). 72 Vps15 contains a protein kinase domain whose function and possible substrates are 73 uncertain, as well as HEAT and WD40 repeats. Atg6 is the yeast ortholog of the human 74 tumor suppressor BECN1, and contains an intrinsically disordered region, a coiled coil, 75 and a BARA domain. Yeast complex I further contains the coiled coil subunit Atg14, and 3 76 is the complex that is involved in autophagy initiation. Yeast complex II contains instead 77 another coiled coil protein, Vps38, and functions in endosome maturation. 78 The yeast complexes are prototypes of two human PI 3-kinase complexes 79 (Volinia et al., 1995), which both contain the common core subunits VPS34 (also known 80 as PI3KC3), VPS15, and BECN1 (Backer, 2008; Funderburk et al., 2010; He and Levine, 81 2010; Wirth et al., 2013) (Fig. 1A). The human cognate of PI3KC3 complex I contains 82 ATG14 (also known as ATG14L or BARKOR) (Itakura et al., 2008; Matsunaga et al., 83 2009; Sun et al., 2008; Zhong et al., 2009). We will refer to this complex as PI3KC3-C1. 84 ATG14 is specifically recruited to sites of autophagosome initiation (Fogel et al., 2013; 85 Ge et al., 2014; Graef et al., 2013; Hamasaki et al., 2013; Matsunaga et al., 2010; Wirth 86 et al., 2013). A second human PI3KC3 contains, in place of ATG14, the UV resistance- 87 associated gene product, UVRAG. We will refer to this complex as PI3KC3-C2. 88 PI3KC3-C2 has been proposed to function at later stages in autophagy (Liang et al., 89 2006), but is not currently thought to participate in the initiation of autophagosome 90 biogenesis. The overall objective of this study was to define the architecture of the two 91 human PI3KC3 complexes. One aspect of the larger goal was to ascertain whether there 92 are gross structural differences between PI3KC3-C1 and –C2 that might affect their 93 relative ability to target the ER and initiate autophagy. 94 The pivotal role of PI3KC3 in the basic mechanism of autophagy induction, and 95 the potential importance of PI3KC3 modulators in treating cancer, neurodegenerative, 96 and other diseases are driving interest in the structure of the complex. To date, crystal 97 structures have been obtained for the catalytic core and helical domains of Drosophila 98 Vps34 (Miller et al., 2010), the WD40 propeller domain of yeast Vps15 (Heenan et al., 99 2009), the coiled coil domain of human BECN1 (Li et al., 2012), and the BARA domains 100 of yeast and human Atg6/BECN1 (Huang et al., 2012; Noda et al., 2012). Structures of 101 homologs of the Vps34 C2 domain and the Vps15 HEAT repeats and protein kinase 102 domains are available. These various regions have to work together in a single complex 103 in autophagy initiation. Currently, there are essentially no data on the structure of the 104 361.8 kDa quaternary assembly of these subunits with each other in PI3KC3-C1 (Hurley 105 and Schulman, 2014). We view this information as essential, if we are to begin to 4 106 understand how autophagy is initiated and how it is regulated, and to address the 107 potential for therapeutic modulation of PI3KC3-C1. 108 To this end, we reconstituted active human PI3KC3-C1 and PI3KC3-C2 by co- 109 expression of all four subunits and imaged the complex by electron microscopy (EM). 110 The complexes are elongated, loosely connected, and dynamic, yet by careful selection of 111 class averages and Bayesian data processing, we were able to generate a three- 112 dimensional reconstruction of PI3KC3-C1 at 28 Å resolution. We have mapped the 113 positions of the various domains of the subunits by tagging with maltose binding protein 114 (MBP). Insight into large-scale conformational fluctuations of PI3KC3-C1 was obtained 115 from EM, while local dynamics was mapped by hydrogen-deuterium exchange. Taken 116 together, the data led us to a model for the subunit architecture and dynamics of PI3KC3- 117 C1. The main architectural principles also hold for the PI3KC3-C2 complex. The 118 structural model suggests that the VPS15 kinase domain acts as a latch to regulate PI 3- 119 kinase activity in both the PI3KC3-C1 and C2. 120 121 Results 122 Reconstitution and imaging of PI3KC3-C1 123 In order to generate sufficient quantities of compositionally homogeneous PI3KC3-C1, 124 synthetic DNA constructs encoding VPS34, VPS15, BECN1 and ATG14 were co- 125 transfected in HEK293 cells. The resulting complex contained all four subunits at 126 apparently equal stoichiometry (Fig. 1B), and the subunits co-migrated as a single peak 127 on gel filtration chromatography (Fig. 2A). The material was enzymatically active as 128 judged by ATP hydrolysis in the presence of PI (Fig. 2B) and by the formation of PI(3)P 129 from PI as assessed by thin layer chromatography (Fig. 1C). ATP hydrolysis by PI3KC3- 130 C1 was essentially completely blocked by the PI 3-kinase inhibitor wortmannin (Fig. 2B ), 131 confirming that the ATPase activity was due to the lipid kinase domain and not either the 132 VPS15 kinase domain or contaminants. 133 PI3KC3-C1 samples were stained with uranyl formate and imaged by EM (Fig. 134 3A). Two-dimensional class averages for PI3KC3-C1 are shown in Fig. 1D. The class 135 averages revealed a V-shaped particle with two arms of approximately 20 nm in length.
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