RESUMEN
The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.
Asunto(s)
Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Unión al GTP rab1/química , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab5/química , Proteínas de Unión al GTP rab5/metabolismo , Beclina-1/química , Beclina-1/genética , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Estructura Secundaria de Proteína , Tomografía , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab5/genéticaRESUMEN
The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is required for the initiation of essentially all macroautophagic processes. PI3KC3-C1 consists of the lipid kinase catalytic subunit VPS34, the VPS15 scaffold, and the regulatory BECN1 and ATG14 subunits. The VPS34 catalytic domain and BECN1:ATG14 subcomplex do not touch, and it is unclear how allosteric signals are transmitted to VPS34. We used EM and crosslinking mass spectrometry to dissect five conformational substates of the complex, including one in which the VPS34 catalytic domain is dislodged from the complex but remains tethered by an intrinsically disordered linker. A "leashed" construct prevented dislodging without interfering with the other conformations, blocked enzyme activity in vitro, and blocked autophagy induction in yeast cells. This pinpoints the dislodging and tethering of the VPS34 catalytic domain, and its regulation by VPS15, as a master allosteric switch in autophagy induction.
Asunto(s)
Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Regulación Alostérica , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/genética , Células HEK293 , Humanos , Espectrometría de Masas/métodos , Mutación , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Relación Estructura-Actividad , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismoRESUMEN
Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.
Asunto(s)
Membrana Celular/enzimología , Fosfatidilinositol 3-Quinasas Clase III/ultraestructura , Endosomas/enzimología , Membrana Celular/química , Fosfatidilinositol 3-Quinasas Clase III/química , Cristalografía por Rayos X , Endosomas/química , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/enzimología , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/ultraestructuraRESUMEN
Autophagy is a tightly controlled degradation process of all eukaryotes. It includes the sequestration of cytoplasmic contents and organelles within a double-membraned autophagosome. Autophagy involves core autophagy related (atg) genes as well as genes regulating vesicle trafficking. Previously, we analyzed the impact of proteins of the core autophagic machinery SmATG7, SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. While deletion of Smatg8 and Smatg4 abolished fruiting-body formation and impaired vegetative growth, Smatg7 is required for viability. In yeast, the phosphatidylinositol 3-kinase vacuolar protein sorting 34 (Vps34) and its myristoylated membrane targeting unit, the protein kinase Vps15 have been shown to be important regulators of autophagy and vacuolar protein sorting. However, their exact role in filamentous ascomycetes remains elusive. To determine the function of Smvps34 and Smvps15 we isolated genes with high sequence similarity to Saccharomyces cerevisiae VPS34 and VPS15. For both genes we were not able to generate a homokaryotic knockout mutant in S. macrospora, suggesting that Smvps34 and Smvps15 are required for viability. Furthermore, we analyzed the repertoire of vps genes encoded by S. macrospora and could identify putative homologs of nearly all of the 61 VPS genes of S. cerevisiae.
