RESUMEN
The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation. Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Hepatocitos/metabolismo , Gotas Lipídicas/metabolismo , Biomarcadores/metabolismo , Línea Celular Tumoral , AMP Cíclico/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Técnicas de Silenciamiento del Gen , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Hepatocitos/ultraestructura , Humanos , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/ultraestructura , Hígado/efectos de los fármacos , Hígado/metabolismo , Ácido Oléico/farmacología , Perilipina-3 , Transporte de Proteínas/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5'-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteína Coat de Complejo I/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Aparato de Golgi/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/antagonistas & inhibidores , Factores de Ribosilacion-ADP/genética , Proteína Coat de Complejo I/genética , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Proteínas Activadoras de GTPasa/genética , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Membranas Intracelulares/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/genética , Factores de Transcripción/metabolismoRESUMEN
We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min. Removal of the PAP inhibitor then results in a rapid burst of buds, vesicles, and tubules that peaks within 2 min. The inability to form buds in the presence of propranolol does not appear to be correlated with a loss of ARFGAP1 from Golgi membranes, as knockdown of ARFGAP1 by RNA interference has little or no effect on actual bud formation. Rather, knockdown of ARFGAP1 results in an increase in membrane buds and a decrease of vesicles and tubules suggesting it functions in the late stages of scission. How DAG promotes bud formation is discussed.
Asunto(s)
Diglicéridos/metabolismo , Aparato de Golgi/metabolismo , Animales , Proteínas Activadoras de GTPasa/metabolismo , Aparato de Golgi/enzimología , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Modelos Biológicos , Fosfatidato Fosfatasa/metabolismo , Ácidos Fosfatidicos/metabolismo , RatasRESUMEN
We have previously uncovered roles for phospholipase D (PLD) and an unknown cytosolic protein in the formation of cytosolic lipid droplets using a cell-free system. In this report, PLD1 has been identified as the relevant isoform, and extracellular signal-regulated kinase 2 (ERK2) as the cytosolic protein. Increased expression of PLD1 increased lipid droplet formation whereas knockdown of PLD1 using siRNA was inhibitory. A role for ERK2 in basal lipid droplet formation was revealed by overexpression or microinjection, and ablation by siRNA knockdown or pharmacological inhibition. Similar manipulations of other Map kinases such as ERK1, JNK1 or JNK2 and p38alpha or p38beta were without effect. Insulin stimulated the formation of lipid droplets and this stimulation was inhibited by knockdown of PLD1 (by siRNA) and by inhibition or knockdown (by siRNA) of ERK2. Inhibition of ERK2 eliminated the effect of PLD1 on lipid droplet formation without affecting PLD1 activity, suggesting that PLD1 functions upstream of ERK2. ERK2 increased the phosphorylation of dynein which increased the amount of the protein on ADRP-containing lipid droplets. Microinjection of antibodies to dynein strongly inhibited the formation of lipid droplets, demonstrating that dynein has a central role in this formation. Thus dynein is a possible target for ERK2.
Asunto(s)
Citosol/metabolismo , Lípidos/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fosfolipasa D/metabolismo , Animales , Anticuerpos/farmacología , Células Cultivadas , Citosol/efectos de los fármacos , Dineínas/antagonistas & inhibidores , Dineínas/metabolismo , Insulina/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Células 3T3 NIH , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/biosíntesis , Fosforilación , ARN Interferente Pequeño/farmacologíaRESUMEN
OBJECTIVE: We investigated the role of adipocyte differentiation-related protein (ADRP) in triglyceride turnover and in the secretion of very low-density lipoprotein (VLDL) from McA-RH7777 cells and primary rat hepatocytes. METHODS AND RESULTS: An increase in the expression of ADRP increased triglyceride accumulation in cytosolic lipid droplets and prevented the incorporation of fatty acids into secretable triglycerides, thereby reducing the secretion of triglycerides as well as of apolipoprotein B-100 (apoB-100) and apoB-48 VLDL. The ability of ADRP to block the secretion of apoB-100 VLDL1 decreased with increasing quantities of fatty acids in the medium, indicating a saturable process and emphasizing the importance of sequestering of fatty acids for the effect of ADRP on VLDL secretion. Knockdown (small interfering RNA) of ADRP decreased the pool of cytosolic lipid droplets but increased only the secretion of apoB-48 VLDL1. Additionally, there was an increased flow of fatty acids into beta-oxidation. CONCLUSIONS: ADRP is essential for the accumulation of triglycerides in cytosolic lipid droplets. An increase in ADRP prevents the formation of VLDL by diverting fatty acids from the VLDL assembly pathway into cytosolic triglycerides, whereas a decrease of the protein increases the sorting of fatty acids to beta-oxidation and promotes the secretion of apoB-48 VLDL1.
Asunto(s)
Citosol/metabolismo , Ácidos Grasos/metabolismo , Lipoproteínas VLDL/antagonistas & inhibidores , Proteínas de la Membrana/fisiología , Triglicéridos/metabolismo , Animales , Apolipoproteína B-48 , Apolipoproteínas B/metabolismo , Línea Celular Tumoral , Técnicas de Transferencia de Gen , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Lipoproteínas VLDL/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Oxidación-Reducción/efectos de los fármacos , Perilipina-2 , ARN Interferente Pequeño/farmacología , RatasRESUMEN
Intracellular transport has remained central to cell biology now for more than 40 years. Despite this, we still lack an overall mechanistic framework that describes transport in different parts of the cell. In the secretory pathway, basic questions, such as how biosynthetic cargo traverses the pathway, are still debated. Historically, emphasis was first put on interpreting function from morphology at the ultrastructural level revealing membrane structures such as the transitional ER, vesicular carriers, vesicular tubular clusters, Golgi cisternae, Golgi stacks and the Golgi ribbon. This emphasis on morphology later switched to biochemistry and yeast genetics yielding many of the key molecular players and their associated functions that we know today. More recently, microscopy studies of living cells incorporating biophysics and system analysis has proven useful and is often used to readdress earlier findings, sometimes with surprising outcomes.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Aparato de Golgi/metabolismo , Animales , Glicosiltransferasas/metabolismo , Transporte de ProteínasRESUMEN
Microsomal triglyceride transfer protein (MTP) is rate-limiting in the assembly and secretion of lipoproteins containing apolipoprotein (apo) B. Previously we demonstrated that Wy 14,643 (Wy), a peroxisome proliferator-activated receptor (PPAR) alpha agonist, increases apoB-100 secretion despite decreased triglyceride synthesis. In this study, we sought to determine whether PPARalpha activation increases MTP expression and activity. Treatment with Wy increased hepatic MTP expression and activity in rats and mice and increased MTP expression in primary cultures of rat and mouse hepatocytes. Addition of actinomycin D blocked this increase and the MTP promoter (-136 to +67) containing a conserved DR1 element was activated by Wy, showing that PPARalpha activates transcription of the gene. Wy did not affect MTP expression in the intestine or in cultured hepatocytes from PPARalpha-null mice. A retinoid X receptor agonist (9-cis-retinoic acid), but not a PPARgamma agonist (rosiglitazone), increased MTP mRNA expression in cultured hepatocytes from both wild type and PPARalpha-null mice. In rat hepatocytes incubated with Wy, MTP mRNA levels increased between 6 and 24 h, and MTP protein expression and apoB-100 secretion increased between 24 and 72 h. In conclusion, PPARalpha activation stimulates hepatic MTP expression via increased transcription of the Mtp gene. This effect is paralleled by a change in apoB-100 secretion, indicating that the effect of Wy on apoB-100 secretion is mediated by increased expression of MTP.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Hígado/metabolismo , PPAR alfa/metabolismo , Alitretinoína , Animales , Apolipoproteínas B/metabolismo , Línea Celular , Células Cultivadas , Dactinomicina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Pirimidinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Rosiglitazona , Tiazolidinedionas/farmacología , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
OBJECTIVE: We investigated the role of ADP ribosylation factor 1 (ARF1) in the assembly of very-low-density lipoproteins (VLDLs). METHODS AND RESULTS: The dominant-negative ARF1 mutant, T31N, decreased the assembly of apoB-100 VLDL 1 (Svedberg floatation units [Sf] 60 to 400) by 80%. The decrease coincided with loss of coatamer I (COPI) from the Golgi apparatus and inhibition of anterograde transport, as demonstrated by time-lapse studies of the vesicular stomatitis virus G protein. The VLDL 1 assembly was also completely inhibited at 15 degrees C. Thus, the antegrade transport is essential for the assembly of VLDL 1. Intracellular localization of N-acetylgalactosaminyl transferase 2 indicated that the Golgi apparatus was at least partly intact when the VLDL assembly was inhibited. Transient transfection with phospholipase D 1 increased the assembly of VLDL 1 and VLDL 2 (Sf 20 to 60). Overexpression of ARF1 in stably transfected McA-RH7777 cells increased the secretion of VLDL 2 but not of VLDL 1, which was dependent on the availability of oleic acid. Secretion of VLDL 1 increased with increasing amounts of oleic acid, and VLDL 2 secretion decreased simultaneously. CONCLUSIONS: Overexpression of ARF1 increased the assembly of VLDL 2 but not of VLDL 1, whose production was dependent on both anterograde transport and the availability of fatty acids.
Asunto(s)
Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Apolipoproteínas B/metabolismo , Animales , Apolipoproteína B-100 , Transporte Biológico , Carcinoma Hepatocelular , Línea Celular Tumoral , Proteína Coat de Complejo I/metabolismo , Expresión Génica , Aparato de Golgi/metabolismo , Lipoproteínas VLDL/metabolismo , Neoplasias Hepáticas , Mutación , Ácido Oléico/farmacocinética , Fosfolipasa D/metabolismo , RatasRESUMEN
We developed a microsome-based, cell-free system that assembles newly formed triglyceride (TG) into spherical lipid droplets. These droplets were recovered in the d
Asunto(s)
Caveolinas/metabolismo , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Fosfolipasa D/metabolismo , Vimentina/metabolismo , Células 3T3 , Adipocitos/metabolismo , Animales , Caveolina 1 , Caveolina 2 , Sistema Libre de Células , Técnicas In Vitro , Ratones , Microscopía Electrónica , Microsomas/metabolismo , Perilipina-2 , Ratas , Triglicéridos/metabolismoRESUMEN
The peroxisome proliferator-activated receptor (PPAR) alpha agonist WY 14,643 increased the secretion of apolipoprotein (apo) B-100, but not that of apoB-48, and decreased triglyceride biosynthesis and secretion from primary rat hepatocytes. These effects resulted in decreased secretion of apoB-100-very low density lipoprotein (VLDL) and an increased secretion of apoB-100 on low density lipoproteins/intermediate density lipoproteins. ApoB-48-VLDL was also replaced by more dense particles. The proteasomal inhibitor lactacystin did not influence the recovery of apoB-100 or apoB-48 in primary rat hepatocytes, indicating that co-translational (proteasomal) degradation is of less importance in these cells. Treatment with WY 14,643 made the recovery of apoB-100 sensitive to lactacystin, most likely reflecting the decreased biosynthesis of triglycerides. The PPAR alpha agonist induced a significant increase in the accumulation of pulse-labeled apoB-100 even after a short pulse (2-5 min). There was also an increase in apoB-100 nascent polypeptides, indicating that the co-translational degradation of apoB-100 was inhibited. However, a minor influence on an early posttranslation degradation cannot be excluded. This decreased co-translational degradation of apoB-100 explained the increased secretion of the protein. The levels of apoB-48 remained unchanged during these pulse-chase experiments, and albumin production was not affected, indicating a specific effect of PPAR alpha agonists on the co-translational degradation of apoB-100. These findings explain the difference in the rate of secretion of the two apoB proteins seen after PPAR alpha activation. PPAR alpha agonists increased the expression and biosynthesis of liver fatty acid-binding protein (LFABP). Increased expression of LFABP by transfection of McA-RH7777 cells increased the secretion of apoB-100, decreased triglyceride biosynthesis and secretion, and increased PPAR alpha mRNA levels. These findings suggest that PPAR alpha and LFABP could interact to amplify the effect of endogenous PPAR alpha agonists on the assembly of VLDL.