RESUMEN
Phosphoinositide (PPI) lipids are a crucial class of low-abundance signaling molecules that regulate many processes within cells. Methods that enable simultaneous detection of all PPI lipid species provide a wholistic snapshot of the PPI profile of cells, which is critical for probing PPI biology. Here we describe a method for the simultaneous measurement of cellular PPI levels by metabolically labeling yeast or mammalian cells with myo-3H-inositol, extracting radiolabeled glycerophosphoinositides, and separating lipid species on an anion exchange column via HPLC.
Asunto(s)
Marcaje Isotópico/métodos , Fosfatos de Fosfatidilinositol/química , Fosfatidilinositoles/análisis , Animales , Fenómenos Bioquímicos , Humanos , Inositol/química , Fosfatidilinositol 3-Quinasas/análisis , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/análisis , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/química , Fosfatidilinositoles/metabolismo , Radioisótopos/química , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], because mutations in PI(3,5)P2-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P2 for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P2 synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P2 in controlling synapse function and/or plasticity. PI(3,5)P2 is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P2 synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P2 being among the most dynamic. The levels of PI(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P2 and reduced synaptic strength following increased PI(3,5)P2 levels. Finally, inhibiting PI(3,5)P2 synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P2 dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P2-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.
Asunto(s)
Depresión Sináptica a Largo Plazo/fisiología , Neuronas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Membranas Sinápticas/metabolismo , Animales , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana , Ratones , Ratones Noqueados , Neuronas/citología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/genética , Transporte de Proteínas , Receptores AMPA/genética , Sinapsis/genética , Membranas Sinápticas/genéticaRESUMEN
TORC1, a conserved protein kinase, regulates cell growth in response to nutrients. Localization of mammalian TORC1 to lysosomes is essential for TORC1 activation. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P(2)), an endosomal signaling lipid, is implicated in insulin-dependent stimulation of TORC1 activity in adipocytes. This raises the question of whether PI(3,5)P(2) is an essential general regulator of TORC1. Moreover, the subcellular location where PI(3,5)P(2) regulates TORC1 was not known. Here we report that PI(3,5)P(2) is required for TORC1 activity in yeast and regulates TORC1 on the vacuole (lysosome). Furthermore, we show that the TORC1 substrate, Sch9 (a homologue of mammalian S6K), is recruited to the vacuole by direct interaction with PI(3,5)P(2), where it is phosphorylated by TORC1. Of importance, we find that PI(3,5)P(2) is required for multiple downstream pathways via TORC1-dependent phosphorylation of additional targets, including Atg13, the modification of which inhibits autophagy, and phosphorylation of Npr1, which releases its inhibitory function and allows nutrient-dependent endocytosis. These findings reveal PI(3,5)P(2) as a general regulator of TORC1 and suggest that PI(3,5)P(2) provides a platform for TORC1 signaling from lysosomes.
Asunto(s)
Alimentos , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Autofagia , Membranas Intracelulares/metabolismo , Modelos Biológicos , Fosforilación , Saccharomyces cerevisiae/citología , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , Vacuolas/metabolismoRESUMEN
Vac8p, an armadillo (ARM) repeat protein, is required for multiple vacuolar processes. It functions in vacuole inheritance, cytoplasm-to-vacuole protein targeting pathway, formation of the nucleus-vacuole junction and vacuole-vacuole fusion. These functions each utilize a distinct Vac8p-binding partner. Here, we report an additional Vac8p function: caffeine resistance. We show that Vac8p function in caffeine resistance is mediated via a newly identified Vac8p-binding partner, Tco89p. The interaction between Vac8p and each binding partner requires an overlapping subset of Vac8p ARM repeats. Moreover, these partners can compete with each other for access to Vac8p. Furthermore, Vac8p is enriched in three separate subdomains on the vacuole, each with a unique binding partner dedicated to a different vacuolar function. These findings suggest that a major role of Vac8p is to spatially separate multiple functions thereby enabling vacuole inheritance to occur concurrently with other vacuolar processes.
Asunto(s)
Cafeína/farmacología , Farmacorresistencia Fúngica/fisiología , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Vacuolas/fisiología , Membrana Celular/metabolismo , Lipoproteínas/genética , Proteínas de la Membrana/genética , Sistemas de Lectura Abierta , Inhibidores de Fosfodiesterasa/farmacología , Unión Proteica , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos Híbridos , Vacuolas/ultraestructura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Phosphoinositide lipids regulate complex events via the recruitment of proteins to a specialized region of the membrane at a specific time. Precise control of both the synthesis and turnover of phosphoinositide lipids is integral to membrane trafficking, signal transduction, and cytoskeletal rearrangements. Little is known about the acute regulation of the levels of these signaling lipids. When Saccharomyces cerevisiae cells are treated with hyperosmotic medium the levels of phosphatidylinositol 3,5-bisphosphate (PI3,5P(2)) increase 20-fold. Here we show that this 20-fold increase is rapid and occurs within 5 min. Surprisingly, these elevated levels are transient. Fifteen minutes following hyperosmotic shock they decrease at a rapid rate, even though the cells remain in hyperosmotic medium. In parallel with the rapid increase in the levels of PI3,5P(2), vacuole volume decreases rapidly. Furthermore, concomitant with a return to basal levels of PI3,5P(2) vacuole volume is restored. We show that Fig 4p, consistent with its proposed role as a PI3,5P(2) 5-phosphatase, is required in vivo for this rapid return to basal levels of PI3,5P(2). Surprisingly, we find that Fig 4p is also required for the hyperosmotic shock-induced increase in PI3,5P(2) levels. These findings demonstrate that following hyperosmotic shock, large, transient changes occur in the levels of PI3,5P(2) and further suggest that Fig 4p is important in regulating both the acute rise and subsequent fall in PI3,5P(2) levels.
Asunto(s)
Flavoproteínas/fisiología , Presión Osmótica , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Membrana Celular/química , Monoéster Fosfórico Hidrolasas , Proteínas de Saccharomyces cerevisiae/análisis , Factores de Tiempo , Regulación hacia ArribaRESUMEN
The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1-Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1-Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1-Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1-Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido , Fusión de Membrana/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Proteínas Portadoras/genética , Sustancias Macromoleculares , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Normal cellular function requires that organelles be positioned in specific locations. The direction in which molecular motors move organelles is based in part on the polarity of microtubules and actin filaments. However, this alone does not determine the intracellular destination of organelles. For example, the yeast class V myosin, Myo2p, moves several organelles to distinct locations during the cell cycle. Thus the movement of each type of Myo2p cargo must be regulated uniquely. Here we report a regulatory mechanism that specifically provides directionality to vacuole movement. The vacuole-specific Myo2p receptor, Vac17p, has a key function in this process. Vac17p binds simultaneously to Myo2p and to Vac8p, a vacuolar membrane protein. The transport complex, Myo2p-Vac17p-Vac8p, moves the vacuole to the bud, and is then disrupted through the degradation of Vac17p. The vacuole is ultimately deposited near the centre of the bud. Removal of a PEST sequence (a potential signal for rapid protein degradation) within Vac17p causes its stabilization and the subsequent 'backward' movement of vacuoles, which mis-targets them to the neck between the mother cell and the bud. Thus the regulated disruption of this transport complex places the vacuole in its proper location. This may be a general mechanism whereby organelles are deposited at their terminal destination.