Cholesterol has important functions in the organization of membrane structure and this may be mediated via the formation of cholesterol-rich, liquid-ordered membrane microdomains often referred to as lipid rafts. Methyl-beta-cyclodextrin (cyclodextrin) is commonly used in cell biology studies to extract cholesterol and therefore disrupt lipid rafts. However, in this study we reassessed this experimental strategy and investigated the effects of cyclodextrin on the physical properties of sonicated and carbonate-treated intracellular membrane vesicles isolated from Cos-7 fibroblasts. We treated these membranes, which mainly originate from the trans -Golgi network and endosomes, with cyclodextrin and measured the effects on their equilibrium buoyant density, protein content, represented by the palmitoylated protein phosphatidylinositol 4-kinase type II α , and cholesterol. Despite the reduction in mass stemming from cholesterol removal, the vesicles became denser, indicating a possible large volumetric decrease, and this was confirmed by measurements of hydrodynamic vesicle size. Subsequent mathematical analyses demonstrated that only half of this change in membrane size was attributable to cholesterol loss. Hence, the non-selective desorption properties of cyclodextrin are also involved in membrane size and density changes. These findings may have implications for preceding studies that interpreted cyclodextrin-induced changes to membrane biochemistry in the context of lipid raft disruption without taking into account our finding that cyclodextrin treatment also reduces membrane size.
Increasing evidence for the organization of cell-surface proteins and lipids into different detergent-insoluble rafts led us to investigate epidermal growth factor (EGF) receptor activation in the plasma membranes of A431 carcinoma cells, using a combination of cell fractionation and immunoprecipitation techniques. Density-gradient centrifugation of sodium carbonate cell extracts revealed that the vast majority of both stimulated and unstimulated EGF receptors were concentrated in a caveolin-rich light membrane (CLM) fraction, with the biochemical characteristics of detergent-insoluble glycolipid-rich domains (DIGs). However, ultrastructural analysis of the CLM fraction revealed that it contained a heterogeneous collection of vesicles, some with sizes greater than that expected for individual caveolae. Experiments with detergent-solubilized cells and isolated CLMs indicated that, in contrast with caveolin, EGF receptors were unlikely to be localized to DIG domains. Furthermore, immunoisolation of caveolin from CLMs revealed that EGF receptor activation occurs in a compartment distinct from caveolae. Similarly, using an anti-(EGF receptor) antibody, the bulk of the cellular caveolin was not co-immunoprecipitated from CLMs, thereby confirming that these two proteins reside in separate membrane domains. The deduction that caveolar signalling and EGF receptor activation occur in separable rafts argues for a multiplicity of signal transduction compartments within the plasma membrane. In addition, by demonstrating that EGF receptor activation is compartmentalized within low-density, non-caveolar regions of the plasma membrane, it is also shown that the co-localization of proteins in a CLM fraction is insufficient to prove caveolar localization.
Rafts are small membrane domains containing discrete subsets of lipids and proteins. Although microscopic raft structures termed ‘caveolae’ were described nearly 50 years ago, the importance of rafts, particularly signalling within rafts, is only beginning to be understood. Our studies focus on receptor-dependent phosphoinositide signalling. Using their characteristic buoyancy in density gradients, we and others found that the epidermal growth factor (EGF) receptor, phosphatidyl-inositol 4-kinase and phosphoinositides are localized within a caveolin-rich fraction of A431 carcinoma cells. We subsequently found that membrane fragments containing the EGF receptor and most cellular phosphoinositides can be separated from caveolae. Consequently, components of EGF-dependent phosphoinositide signalling localize to one or more novel types of raft, the composition of which we are currently determining. A key component is the type II phosphatidylinositol 4-kinase, which, for many years, has proven difficult to purify and clone. We describe our recent purification from rafts and cloning of this elusive enzyme, and discuss how the structure sheds light on the rafting of this enzyme.
Abstract The Covid‐19 pandemic caused by the novel Sars‐CoV‐2 coronavirus, has resulted in millions of deaths and disruption to daily life across the globe. University students have been additionally affected by a sudden move to online learning, the closure of campuses and dramatic societal changes that have upended their experiences of higher education. Here we focus on the physical and mental health consequences of the pandemic for this population sector during 2020, and the interdependencies of these impacts. We survey the challenges for infection control on campuses and for monitoring the disease dynamics in student communities. Finally, we explore the psychological and mental health problems that have been exacerbated by the pandemic and evaluate the underlying factors that are most relevant to students.
Phosphatidylinositol (PI) is a phospholipid molecule required for the generation of seven different phosphoinositide lipids which have a diverse range of signaling and trafficking functions. The precise mechanism of phosphatidylinositol supply during receptor activated signaling and the cellular compartmentation of the synthetic process are still incompletely understood and remain controversial despite several decades of research in this area. The synthesis of phosphatidylinositol requires the activity of an enzyme called phosphatidylinositol synthase, also known as CDIPT, which catalyzes a reversible headgroup exchange reaction on its substrate liponucleotide CDP-diacylglycerol resulting in the incorporation of inositol to generate phosphatidylinositol and the release of CMP. This protocol describes a method for locating PI synthase activity in isolated, intact biological membranes and vesicles.
Type II phosphatidylinositol 4-kinase IIα (PI4KIIα) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KIIα has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KIIα mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KIIα activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KIIα to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fluorescent protein (eGFP)-tagged PI4KIIα demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KIIα was assessed by fluorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KIIα. Further measurements revealed that the reduction in the mobile fraction of PI4KIIα correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion. Type II phosphatidylinositol 4-kinase IIα (PI4KIIα) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KIIα has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KIIα mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KIIα activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KIIα to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fluorescent protein (eGFP)-tagged PI4KIIα demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KIIα was assessed by fluorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KIIα. Further measurements revealed that the reduction in the mobile fraction of PI4KIIα correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion. Phosphatidylinositol 4-phosphate (PI4P) is generated by phosphorylation of phosphatidylinositol (PI) on the 4-position by phosphatidylinositol 4-kinases (1.Balla A. Balla T. Phosphatidylinositol 4-kinases: old enzymes with emerging functions.Trends Cell Biol. 2006; 16: 351-361Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). In mammalian cells (2.Simons J.P. Al-Shawi R. Minogue S. Waugh M.G. Wiedemann C. Evangelou S. Loesch A. Sihra T.S. King R. Warner T.T. et al.Loss of phosphatidylinositol 4-kinase 2alpha activity causes late onset degeneration of spinal cord axons.Proc. Natl. Acad. Sci. USA. 2009; 106: 11535-11539Crossref PubMed Scopus (56) Google Scholar, 3.Wang Y.J. Wang J. Sun H.Q. Martinez M. Sun Y.X. Macia E. Kirchhausen T. Albanesi J.P. Roth M.G. Yin H.L. Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi.Cell. 2003; 114: 299-310Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar), PI4P synthesis is predominately accounted for by the type II phosphatidylinositol 4-kinase IIα isoform (PI4KIIα). Our most recent work has shown that loss of PI4KIIα activity in mice leads to late-onset neurodegeneration (2.Simons J.P. Al-Shawi R. Minogue S. Waugh M.G. Wiedemann C. Evangelou S. Loesch A. Sihra T.S. King R. Warner T.T. et al.Loss of phosphatidylinositol 4-kinase 2alpha activity causes late onset degeneration of spinal cord axons.Proc. Natl. Acad. Sci. USA. 2009; 106: 11535-11539Crossref PubMed Scopus (56) Google Scholar). In addition, Li et al. (4.Li J. Lu Y. Zhang J. Kang H. Qin Z. Chen C. PI4KIIalpha is a novel regulator of tumor growth by its action on angiogenesis and HIF-1alpha regulation.Oncogene. 2010; 29: 2550-2559Crossref PubMed Scopus (45) Google Scholar) have demonstrated that PI4KIIα is overexpressed in a wide range of common cancers where it has a key role in promoting angiogenesis. However, despite its emerging importance in disease, little is known about endogenous factors that regulate PI4KIIα, and there are currently no pharmacological reagents available to modulate its activity in cells. We previously demonstrated that PI4KIIα activity is sensitive to membrane cholesterol levels (5.Waugh M.G. Minogue S. Chotai D. Berditchevski F. Hsuan J.J. Lipid and peptide control of phosphatidylinositol 4-kinase IIalpha activity on Golgi-endosomal rafts.J. Biol. Chem. 2006; 281: 3757-3763Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Given the significant role of the enzyme in major pathologies, we sought to investigate the biochemical and biophysical mechanisms that underlie sterol-sensitive PI4P synthesis. One possible mechanism through which cholesterol could affect PI4KIIα is by modulating its membrane mobility (6.Owen D.M. Williamson D. Rentero C. Gaus K. Quantitative microscopy: protein dynamics and membrane organisation.Traffic. 2009; 10: 962-971Crossref PubMed Scopus (119) Google Scholar, 7.Adkins E.M. Samuvel D.J. Fog J.U. Eriksen J. Jayanthi L.D. Vaegter C.B. Ramamoorthy S. Gether U. Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching.Biochemistry. 2007; 46: 10484-10497Crossref PubMed Scopus (113) Google Scholar, 8.Baier C.J. Gallegos C.E. Levi V. Barrantes F.J. 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Quantitative microscopy: protein dynamics and membrane organisation.Traffic. 2009; 10: 962-971Crossref PubMed Scopus (119) Google Scholar), and in the case of an enzyme such as PI4KIIα, this is likely to be important for the kinetics of product formation. However, it is important to point out that nothing is known about the membrane dynamics of PI4KIIα. Membrane lipid composition and, in particular, membrane cholesterol concentration underlie biophysical parameters such as membrane fluidity, viscosity, and geometry, all of which are known to modulate membrane protein mobility (6.Owen D.M. Williamson D. Rentero C. Gaus K. Quantitative microscopy: protein dynamics and membrane organisation.Traffic. 2009; 10: 962-971Crossref PubMed Scopus (119) Google Scholar). Cholesterol also has a role in the formation and organization of cholesterol-rich microdomains, often referred to as lipid rafts (11.Lingwood D. Kaiser H.J. Levental I. Simons K. 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Differential accumulation of cholesterol in Golgi compartments of normal and Niemann-Pick type C fibroblasts incubated with LDL: a cytochemical freeze-fracture study.J. Lipid Res. 1993; 34: 1165-1176Abstract Full Text PDF PubMed Google Scholar, 15.Orci L. Montesano R. Meda P. Malaisse-Lagae F. Brown D. Perrelet A. Vassalli P. Heterogeneous distribution of filipin—cholesterol complexes across the cisternae of the Golgi apparatus.Proc. Natl. Acad. Sci. USA. 1981; 78: 293-297Crossref PubMed Scopus (196) Google Scholar, 16.van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids: where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Crossref PubMed Scopus (4618) Google Scholar) and also contains high levels of PI4P (17.Balla A. Tuymetova G. Tsiomenko A. Varnai P. Balla T. A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1.Mol. 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The role of the phosphoinositides at the Golgi complex.Biochim. Biophys. Acta. 2005; 1744: 396-405Crossref PubMed Scopus (115) Google Scholar, 22.Di Paolo G. De Camilli P. Phosphoinositides in cell regulation and membrane dynamics.Nature. 2006; 443: 651-657Crossref PubMed Scopus (2104) Google Scholar). At the TGN, PI4KIIα activity is required for clathrin-dependent vesicle formation (3.Wang Y.J. Wang J. Sun H.Q. Martinez M. Sun Y.X. Macia E. Kirchhausen T. Albanesi J.P. Roth M.G. Yin H.L. Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi.Cell. 2003; 114: 299-310Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 23.Wang J. Sun H.Q. Macia E. Kirchhausen T. Watson H. Bonifacino J.S. Yin H.L. PI4P promotes the recruitment of the GGA adaptor proteins to the trans-Golgi network and regulates their recognition of the ubiquitin sorting signal.Mol. Biol. Cell. 2007; 18: 2646-2655Crossref PubMed Scopus (129) Google Scholar). 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Cell Sci. 2007; 120: 3640-3652Crossref PubMed Scopus (29) Google Scholar). The TGN pool of PI4KIIα corresponds to the juxtanuclear and vesicular PI4KIIα pool that colocalizes with the TGN proteins syntaxin-6 (33.Bock J.B. Klumperman J. Davanger S. Scheller R.H. Syntaxin 6 functions in trans-Golgi network vesicle trafficking.Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar) and TGN46 (34.Banting G. Ponnambalam S. TGN38 and its orthologues: roles in post-TGN vesicle formation and maintenance of TGN morphology.Biochim. Biophys. Acta. 1997; 1355: 209-217Crossref PubMed Scopus (98) Google Scholar) both by microscopy (20.Weixel K.M. Blumental-Perry A. Watkins S.C. Aridor M. Weisz O.A. Distinct Golgi populations of phosphatidylinositol 4-phosphate regulated by phosphatidylinositol 4-kinases.J. Biol. Chem. 2005; 280: 10501-10508Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 31.Waugh M.G. Minogue S. Blumenkrantz D. Anderson J.S. Hsuan J.J. Identification and characterization of differentially active pools of type IIalpha phosphatidylinositol 4-kinase activity in unstimulated A431 cells.Biochem. J. 2003; 376: 497-503Crossref PubMed Scopus (25) Google Scholar, 35.Barylko B. Mao Y.S. Wlodarski P. Jung G. Binns D.D. Sun H.Q. Yin H.L. Albanesi J.P. Palmitoylation controls the catalytic activity and subcellular distribution of phosphatidylinositol 4-kinase II{alpha}.J. Biol. Chem. 2009; 284: 9994-10003Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and by subcellular fractionation (36.Waugh M.G. Minogue S. Anderson J.S. Balinger A. Blumenkrantz D. Calnan D.P. Cramer R. Hsuan J.J. Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum.Biochem. J. 2003; 373: 57-63Crossref PubMed Scopus (53) Google Scholar). PI4KIIα is unique among all the PI kinases in that it is constitutively associated with TGN membrane domains (20.Weixel K.M. Blumental-Perry A. Watkins S.C. Aridor M. Weisz O.A. Distinct Golgi populations of phosphatidylinositol 4-phosphate regulated by phosphatidylinositol 4-kinases.J. Biol. Chem. 2005; 280: 10501-10508Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 35.Barylko B. Mao Y.S. Wlodarski P. Jung G. Binns D.D. Sun H.Q. Yin H.L. Albanesi J.P. Palmitoylation controls the catalytic activity and subcellular distribution of phosphatidylinositol 4-kinase II{alpha}.J. Biol. Chem. 2009; 284: 9994-10003Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 36.Waugh M.G. Minogue S. Anderson J.S. Balinger A. Blumenkrantz D. Calnan D.P. Cramer R. Hsuan J.J. Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum.Biochem. 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Wang J. Wlodarski P. Barylko B. Binns D.D. Shu H. Yin H.L. Albanesi J.P. Molecular determinants of activation and membrane targeting of phosphoinositol 4-kinase IIbeta.Biochem. J. 2008; 409: 501-509Crossref PubMed Scopus (31) Google Scholar). PI4KIIα palmitoylation is essential for both kinase activity and for localization to the TGN, but this modification is not absolutely required for membrane association of the enzyme (35.Barylko B. Mao Y.S. Wlodarski P. Jung G. Binns D.D. Sun H.Q. Yin H.L. Albanesi J.P. Palmitoylation controls the catalytic activity and subcellular distribution of phosphatidylinositol 4-kinase II{alpha}.J. Biol. Chem. 2009; 284: 9994-10003Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 38.Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. A novel family of phosphatidylinositol 4-kinases conserved from yeast to humans.J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 39.Jung G. Wang J. Wlodarski P. Barylko B. Binns D.D. Shu H. Yin H.L. Albanesi J.P. Molecular determinants of activation and membrane targeting of phosphoinositol 4-kinase IIbeta.Biochem. J. 2008; 409: 501-509Crossref PubMed Scopus (31) Google Scholar). Here we focus on regulation of PI4KIIα at the TGN, with particular emphasis on the relationship between membrane organization and lipid composition in regulating PI4KIIα dynamics and activity. Previously, we established that PI4KIIα and its phospholipid PI substrate associate with buoyant, TGN membrane domains (36.Waugh M.G. Minogue S. Anderson J.S. Balinger A. Blumenkrantz D. Calnan D.P. Cramer R. Hsuan J.J. Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum.Biochem. 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J. 2003; 373: 57-63Crossref PubMed Scopus (53) Google Scholar). Furthermore, we found that PI4P synthesis in these membrane preparations was sensitive to the manipulation of membrane sterol levels by methyl-β-cyclodextrin (MβCD) (5.Waugh M.G. Minogue S. Chotai D. Berditchevski F. Hsuan J.J. Lipid and peptide control of phosphatidylinositol 4-kinase IIalpha activity on Golgi-endosomal rafts.J. Biol. Chem. 2006; 281: 3757-3763Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Cholesterol sequestration by MβCD is often exploited experimentally to disrupt lipid rafts (reviewed in Ref. 40.Zidovetzki R. Levitan I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies.Biochim. Biophys. Acta. 2007; 1768: 1311-1324Crossref PubMed Scopus (832) Google Scholar). Using this approach, other groups have shown that agonist-sensitive PI4P pools are MβCD-sensitive (41.Naveen B. Shankar B.S. Subrahmanyam G. FcepsilonRI cross-linking activates a type II phosphatidylinositol 4-kinase in RBL 2H3 cells.Mol. Immunol. 2005; 42: 1541-1549Crossref PubMed Scopus (15) Google Scholar) and that MβCD delocalizes raft-associated PI4P (42.Pike L.J. Miller J.M. Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover.J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). In addition to lipid raft disruption, cholesterol removal with MβCD is known to induce a more condensed Golgi morphology (43.Stuven E. Porat A. Shimron F. Fass E. Kaloyanova D. Brugger B. Wieland F.T. Elazar Z. Helms J.B. Intra-Golgi protein transport depends on a cholesterol balance in the lipid membrane.J. Biol. Chem. 2003; 278: 53112-53122Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), partial vesicularisation of TGN membranes (24.Hansen G.H. Niels-Christiansen L.L. Thorsen E. Immerdal L. Danielsen E.M. Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking.J. Biol. Chem. 2000; 275: 5136-5142Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 44.Grimmer S. Iversen T.G. van Deurs B. Sandvig K. Endosome to Golgi transport of ricin is regulated by cholesterol.Mol. Biol. Cell. 2000; 11: 4205-4216Crossref PubMed Scopus (82) Google Scholar), and changes in the lateral mobility of some TGN-associated proteins (45.Lebreton S. Paladino S. Zurzolo C. Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells.J. Biol. Chem. 2008; 283: 29545-29553Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, it is not known how factors, such as membrane domain heterogeneity, TGN membrane continuity, or possible changes to PI4KIIα lateral diffusion, might contribute to the cholesterol-sensitivity of the enzyme. Therefore, in this study we utilize a range of biochemical approaches together with spot photobleaching of enhanced green fluorescent protein (eGFP)-tagged PI4KIIα to elucidate the mechanism underlying cholesterol-dependent PI4P synthesis. Protease inhibitor cocktail tablets (Complete™, without EDTA) were from Roche Diagnostics. Mastoparan was purchased from Calbiochem (Nottingham, UK). MβCD, PI purified from bovine liver, and all the sterols used were bought from Sigma-Aldrich (Poole, Dorset, UK). DMEM, fetal bovine serum, and penicillin/streptomycin were purchased from Invitrogen (Paisley, UK). [γ-32P]ATP (4500–6000 Ci/mmol) was purchased from GE Healthcare. Anti-syntaxin-6 was purchased from BD Pharmingen. Anti-TGN46 was bought from Novus Biologicals. Enantiomeric cholesterol was prepared by catalytic hydrogenation of an enantiomeric desmosterol precursor as described previously (46.Westover E.J. Covey D.F. Brockman H.L. Brown R.E. Pike L.J. Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. Studies with cholesterol enantiomers.J. Biol. Chem. 2003; 278: 51125-51133Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Cells were maintained at 37°C in a humidified incubator at 10% CO2. Cells were cultured in DMEM supplemented with Glutamax, 10% fetal calf serum, 50 i.u./ml penicillin and 50 μg/ml streptomycin. For fluorescence recovery after photobleaching (FRAP) measurements, cells were transfected 24 h prior to the experiments with eGFP-PI4KIIα. Post-nuclear supernatants prepared from confluent cell monolayers were fractionated on a 10–40% w/v sucrose density gradient as previously described (5.Waugh M.G. Minogue S. Chotai D. Berditchevski F. Hsuan J.J. Lipid and peptide control of phosphatidylinositol 4-kinase IIalpha activity on Golgi-endosomal rafts.J. Biol. Chem. 2006; 281: 3757-3763Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 31.Waugh M.G. Minogue S. Blumenkrantz D. Anderson J.S. Hsuan J.J. Identification and characterization of differentially active pools of type IIalpha phosphatidylinositol 4-kinase activity in unstimulated A431 cells.Biochem. J. 2003; 376: 497-503Crossref PubMed Scopus (25) Google Scholar, 36.Waugh M.G. Minogue S. Anderson J.S. Balinger A. Blumenkrantz D. Calnan D.P. Cramer R. Hsuan J.J. Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum.Biochem. J. 2003; 373: 57-63Crossref PubMed Scopus (53) Google Scholar). Buoyant and TGN-enriched membrane fractions 9-10 containing high activity PI4KIIα were harvested as before (5.Waugh M.G. Minogue S. Chotai D. Berditchevski F. Hsuan J.J. Lipid and peptide control of phosphatidylinositol 4-kinase IIalpha activity on Golgi-endosomal rafts.J. Biol. Chem. 2006; 281: 3757-3763Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 31.Waugh M.G. Minogue S. Blumenkrantz D. Anderson J.S. Hsuan J.J. Identification and characterization of differentially active pools of type IIalpha phosphatidylinositol 4-kinase activity in unstimulated A431 cells.Biochem. J. 2003; 376: 497-503Crossref PubMed Scopus (25) Google Scholar). MβCD (20 mM) was added to an equal volume of TGN membranes (usually 1 ml) on ice for 20 min to give a final MβCD concentration of 10 mM. Then 200 μl of sodium carbonate 1M (pH 11.0) was added. The carbonate-treated membranes were probe sonicated followed by adjustment to 40% sucrose w/v in Tris-HCl 10 mM, EDTA 1 mM, and EGTA 1 mM (pH 7.4) to a final volume of 4 ml. A discontinuous sucrose gradient was formed by overlaying the 40% sucrose layer with 4 ml of sucrose 30% w/v and 4 ml sucrose 5% w/v in Tris-HCl 10 mM, EDTA 1 mM, and EGTA 1 mM (pH 7.4). The gradient was centrifuged overnight at 185,000 g at 4°C and 1 ml fractions were harvested beginning at the top of the tube. Equal volume aliquots of density gradient fractions were separated by SDS-PAGE, transferred to PVDF, and probed with anti-PI4KIIα or anti-syntaxin-6 antibodies. Western blots were quantified using image analysis software in Adobe Photoshop CS4. The cholesterol content of equal volume membrane fractions was assayed using the Amplex red cholesterol assay kit (Molecular Probes). PI 4-kinase assays using either endogenous membrane- associated PI or exogenous PI, and add-back of MβCD complexed sterols were performed as previously described (5.Waugh M.G. Minogue S.
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