RESUMEN
In eukaryotes, organelles and vesicles modulate their contents and identities through highly regulated membrane fusion events. Membrane trafficking and fusion are carried out through a series of stages that lead to the formation of SNARE complexes between cellular compartment membranes to trigger fusion. Although the protein catalysts of membrane fusion are well characterized, their response to their surrounding microenvironment, provided by the lipid composition of the membrane, remains to be fully understood. Membranes are composed of bulk lipids (e.g., phosphatidylcholine), as well as regulatory lipids that undergo constant modifications by kinases, phosphatases, and lipases. These lipids include phosphoinositides, diacylglycerol, phosphatidic acid, and cholesterol/ergosterol. Here we describe the roles of these lipids throughout the stages of yeast vacuole homotypic fusion.
Asunto(s)
Colesterol/metabolismo , Ergosterol/metabolismo , Glicéridos/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfatidilinositoles/metabolismo , Vacuolas/metabolismo , Colesterol/química , Ergosterol/química , Glicéridos/química , Humanos , Fusión de Membrana , Ácidos Fosfatidicos/química , Fosfatidilinositoles/química , Vacuolas/químicaRESUMEN
The transport of Ca2+ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca2+ stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol-3-phosphate (PI3P) by the PI3P-5-kinase Fab1 to produce transient PI(3,5)P2 pools. Ca2+ is also released during vacuole fusion upon trans-SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P2 on Ca2+ flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P2 were required for Ca2+ uptake into the vacuole, increased concentrations abolished Ca2+ efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P2 or increased endogenous production of by the hyperactive fab1T2250A mutant. In contrast, the lack of PI(3,5)P2 on vacuoles from the kinase dead fab1EEE mutant showed delayed and decreased Ca2+ uptake. The effects of PI(3,5)P2 were linked to the Ca2+ pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P2 . Experiments with Verapamil inhibited Ca2+ uptake when added at the start of the assay, while adding it after Ca2+ had been taken up resulted in the rapid expulsion of Ca2+ . Vacuoles lacking both Pmc1 and the H+ /Ca2+ exchanger Vcx1 lacked the ability to take up Ca2+ and instead expelled it upon the addition of ATP. Together these data suggest that a balance of efflux and uptake compete during the fusion pathway and that the levels of PI(3,5)P2 can modulate which path predominates.
Asunto(s)
Fosfatos de Fosfatidilinositol , Fosfotransferasas (Aceptor de Grupo Alcohol) , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Adenosina Trifosfatasas , Fosfatidilinositoles , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismoRESUMEN
The accumulation of copper in organisms can lead to altered functions of various pathways and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg+ , Cd2+ and Cu2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu2+ to Cu+ . Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V-ATPase H+ pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that Cu2+ is responsible for the inhibition of vacuole fusion and V-ATPase function. This notion is supported by the differential effects of chelators. The Cu2+ -specific chelator triethylenetetramine rescued fusion, whereas the Cu+ -specific chelator bathocuproine disulfonate had no effect on the inhibited fusion.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cobre/metabolismo , Fusión de Membrana/fisiología , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Ácidos Fosfatidicos/farmacología , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfato/metabolismo , Simulación de Dinámica Molecular , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosforilación , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína/efectos de los fármacos , Proteínas SNARE/química , Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.
Asunto(s)
Adenosina Trifosfatasas/genética , Etilmaleimida/metabolismo , Ácidos Fosfatidicos/química , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequeñas/química , Proteínas de Transporte Vesicular/genética , ATPasas Asociadas con Actividades Celulares Diversas/química , ATPasas Asociadas con Actividades Celulares Diversas/genética , Adenosina Trifosfatasas/química , Fusión de Membrana/efectos de los fármacos , Fusión de Membrana/genética , Lípidos de la Membrana/química , Lípidos de la Membrana/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Simulación de Dinámica Molecular , Proteínas Sensibles a N-Etilmaleimida/química , Proteínas Sensibles a N-Etilmaleimida/genética , Ácidos Fosfatidicos/antagonistas & inhibidores , Proteínas SNARE/química , Proteínas SNARE/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/química , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/genética , Vacuolas/genética , Proteínas de Transporte Vesicular/químicaRESUMEN
Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P2 , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1 Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1 ; however, deleting both genes did not rescue the pah1 Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Fluidez de la Membrana/fisiología , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Endosomas/metabolismo , Fusión de Membrana/fisiología , Fosfatidato Fosfatasa/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica/fisiología , Proteínas SNARE/metabolismo , VacuolasRESUMEN
Yeast vacuole fusion requires the activation of cis-SNARE complexes through priming carried out by Sec18p/N-ethylmaleimide sensitive factor and Sec17p/α-SNAP. The association of Sec18p with vacuolar cis-SNAREs is regulated in part by phosphatidic acid (PA) phosphatase production of diacylglycerol (DAG). Inhibition of PA phosphatase activity blocks the transfer of membrane-associated Sec18p to SNAREs. Thus, we hypothesized that Sec18p associates with PA-rich membrane microdomains before transferring to cis-SNARE complexes upon PA phosphatase activity. Here, we examined the direct binding of Sec18p to liposomes containing PA or DAG. We found that Sec18p preferentially bound to liposomes containing PA compared with those containing DAG by approximately fivefold. Additionally, using a specific PA-binding domain blocked Sec18p binding to PA-liposomes and displaced endogenous Sec18p from isolated vacuoles. Moreover, the direct addition of excess PA blocked the priming activity of isolated vacuoles in a manner similar to chemically inhibiting PA phosphatase activity. These data suggest that the conversion of PA to DAG facilitates the recruitment of Sec18p to cis-SNAREs. Purified vacuoles from yeast lacking the PA phosphatase Pah1p showed reduced Sec18p association with cis-SNAREs and complementation with plasmid-encoded PAH1 or recombinant Pah1p restored the interaction. Taken together, this demonstrates that regulating PA concentrations by Pah1p activity controls SNARE priming by Sec18p.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Membranas Intracelulares/metabolismo , Fusión de Membrana/fisiología , Ácidos Fosfatidicos/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Transporte de ProteínasRESUMEN
The yeast vacuole requires four SNAREs to trigger membrane fusion including the soluble Qc-SNARE Vam7. The N-terminal PX domain of Vam7 binds to the lipid phosphatidylinositol 3-phosphate (PI3P) and the tethering complex HOPS (homotypic fusion and vacuole protein sorting complex), whereas the C-terminal SNARE motif forms SNARE complexes. Vam7 also contains an uncharacterized middle domain that is predicted to be a coiled-coil domain with multiple helices. One helix contains a polybasic region (PBR) composed of Arg-164, Arg-168, Lys-172, Lys-175, Arg-179, and Lys-186. Polybasic regions are often associated with nonspecific binding to acidic phospholipids including phosphoinositides. Although the PX (phox homology) domain alone binds PI3P, we theorized that the Vam7 PBR could bind to additional acidic phospholipids enriched at fusion sites. Mutating each of the basic residues in the PBR to an alanine (Vam7-6A) led to attenuated vacuole fusion. The defective fusion of Vam7-6A was due in part to inefficient association with its cognate SNAREs and HOPS, yet the overall vacuole association of Vam7-6A was similar to wild type. Experiments testing the binding of Vam7 to specific signaling lipids showed that mutating the PBR to alanines augmented binding to PI3P. The increased binding to PI3P by Vam7-6A likely contributed to the observed wild type levels of vacuole association, whereas protein-protein interactions were diminished. PI3P binding was inhibited when the PX domain mutant Y42A was introduced into Vam7-6A to make Vam7-7A. Thus the Vam7 PBR affects PI3P binding by the PX domain and in turn affects binding to SNAREs and HOPS to support efficient fusion.
Asunto(s)
Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Vacuolas/metabolismo , Sustitución de Aminoácidos , Mutación Missense , Fosfatos de Fosfatidilinositol/genética , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína 25 Asociada a Sinaptosomas/genética , Vacuolas/genéticaRESUMEN
Studies examining the relationship between cellular sortilin and VLDL-B100 secretion demonstrate inconsistent results. Current studies explore the possibility that discrepancies may be related to insulin sensitivity. McArdle RH7777 cells (McA cells) cultured under serum enriched conditions lose sensitivity to insulin. Following incubation in serum-free DMEM containing 1% BSA, McA cells become insulin responsive and demonstrate reduced apo B secretion. Current studies indicate that insulin sensitive McA cells express lower cellular sortilin that corresponds with reduction in VLDL-B100 secretion without changes in mRNA of either sortilin or apo B. When sortilin expression is further reduced by siRNA knockdown (KD), there are additional decreases in VLDL-B100 secretion. A crystal structure of human sortilin (hsortilin) identifies two binding sites on the luminal domain for the N- and C-termini of neurotensin (NT). A small organic compound (cpd984) was identified that has strong theoretical binding to the N-terminal site. Both cpd984 and NT bind hsortilin by surface plasmon resonance. In incubations with insulin sensitive McA cells, cpd984 was shown to enhance VLDL-B100 secretion at each level of sortilin KD suggesting cpd984 acted through sortilin in mediating its effect. Current results support a role for sortilin to facilitate VLDL-B100 secretion which is limited to insulin sensitive McA cells. Inconsistent reports of the relationship between VLDL-B100 secretion and sortilin in previous studies may relate to differing functions of sortilin in VLDL-B100 secretion depending upon insulin sensitivity.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Apolipoproteína B-100/metabolismo , Resistencia a la Insulina , Insulina/metabolismo , Lipoproteínas VLDL/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Sitios de Unión , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Simulación del Acoplamiento Molecular , Ratas Sprague-DawleyRESUMEN
BACKGROUND: To evaluate the relationship between sleep and next-day physical activity (PA) under free-living conditions in women. METHODS: Sleep and PA were measured objectively for 7 consecutive days by accelerometry in 330 young adult women (aged 17-25 y). A structural equation model was used to evaluate the relationship between the driving factor of sleep (total sleep or morning wake time) and the amount of nonsleep sedentary (SED) and moderate to vigorous physical activity (MVPA) each day. RESULTS: With sleep duration as the driving factor, the estimates of ßSED and ßMVPA were -0.415 and -0.093, respectively (P ≤ .05). For every hour slept, a 24.9-minute reduction in SED time and a 5.58-minute reduction in MVPA were observed. With wake time as the driving factor, the estimates of ßSED and ßMVPA were -0.636 and -0.149, respectively. For every wake time that was 1 hour later, a 38.2-minute decrease in SED and a 8.9-minute decrease in MVPA (P ≤ .05) were observed. CONCLUSIONS: Women who wake later or who sleep longer tend to get less MVPA throughout the day. Getting up earlier and going to bed earlier may support behaviors that improve PA and lifestyle.
Asunto(s)
Ejercicio Físico , Conducta Sedentaria , Acelerometría , Femenino , Humanos , Estilo de Vida , Sueño , Adulto JovenRESUMEN
Protein-lipid binding interactions play a key role in the regulation of peripheral membrane protein function. Liposome-binding assays are a simple and affordable means of screening for specific protein-lipid interactions. Liposomes are prepared by mixing phospholipid combinations of interest before drying and rehydration. Sonication of the lipid mixture produces small unilamellar vesicles (SUVs) which are incubated with a protein of interest to allow for any binding to occur. Liposomes and liposome-protein complexes are floated on a sucrose gradient by centrifugation to separate them from unbound protein. Bound protein levels are easily determined by SDS-PAGE and Western blotting. This approach provides a reliable means of assaying novel protein-lipid interactions in vitro. Here we use liposome floatation to show the binding of the SNARE-activating protein Sec18 (mammalian NSF) to phosphatidic acid.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Liposomas/metabolismo , Fosfolípidos/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/aislamiento & purificación , Liposomas/química , Fusión de Membrana , Fosfolípidos/química , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/aislamiento & purificaciónRESUMEN
Phosphoinositides (PIs) regulate a myriad of cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) on vacuole fusion remains unknown. PI(3,5)P2 is made by the PI3P 5-kinase Fab1 and has been characterized as a regulator of vacuole fission during hyperosmotic shock, where it interacts with the TRP Ca2+ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5)P2 abolishes homotypic vacuole fusion. This effect was not linked to Yvc1, as fusion was equally affected using yvc1Δ vacuoles. Thus, the effects of C8-PI(3,5)P2 on fusion and fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P2 inhibited vacuole fusion after trans-SNARE pairing. Although SNARE complex formation was unaffected, we found that C8-PI(3,5)P2 blocked outer leaflet lipid mixing. Overproduction of endogenous PI(3,5)P2 by the fab1T2250A hyperactive kinase mutant also inhibited the lipid mixing stage, bolstering the model in which PI(3,5)P2 inhibits fusion when present at elevated levels. Taken together, this work identifies a novel function for PI(3,5)P2 as a regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.