Lipid rafts,caveolae, caveolin-1, and entry by Chlamydiae into host cells

Obligate intracellular bacterial pathogens of the genus Chlamydia are reported to enter host cells by both clathrin-dependent and clathrin-independent processes. C. trachomatis serovar K recently was shown to enter cells via caveolae-like lipid raft domains. We asked here how widespread raft-mediated entry might be among the Chlamydia. We show that C. pneumoniae, an important cause of respiratory infections in humans that additionally is associated with cardiovascular disease, and C. psittaci, an important pathogen in domestic mammals and birds that also infects humans, each enter host cells via cholesterol-rich lipid raft microdomains. Further, we show that C. trachomatis serovars E and F also use these domains to enter host cells. The involvement of these membrane domains in the entry of these organisms was indicated by the sensitivity of their entry to the raft-disrupting agents Nystatin and filipin, and by their intracellular association with caveolin-1, a 22-kDa protein associated with the formation of caveolae in rafts. In contrast, caveolin-marked lipid raft domains do not mediate entry of C. trachomatis serovars A, 36B, and C, nor of LGV serovar L2 and MoPn. Finally, we show that entry of each of these chlamydial strains is independent of cellular expression of caveolin-1. Thus, entry via the Nystatin and filipin-sensitive pathway is dependent on lipid rafts containing cholesterol, rather than invaginated caveolae per se.     

The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion

Chlamydiae replicate within an intracellular vacuole, termed an inclusion, that is non-fusogenic with vesicles of the endosomal or lysosomal compartments. Instead, the inclusion appears to intersect an exocytic pathway from which chlamydiae intercept sphingomyelin en route from the Golgi apparatus to the plasma membrane. Chlamydial protein synthesis is required to establish this interaction. In an effort to identify those chlamydial proteins controlling vesicle fusion, we have prepared polyclonal antibodies against several Chlamydia trachomatis inclusion membrane proteins. Microinjection of polyclonal antibodies against three C. trachomatis inclusion membrane proteins, IncA, F and G, into the cytosol of cells infected with C. trachomatis demonstrates reactivity with antigens on the cytoplasmic face of the inclusion membrane, without apparent inhibition of chlamydial multiplication. Microinjection of antibodies against the C. trachomatis IncA protein, however, results in the development of an aberrant multilobed inclusion structure remarkably similar to that of C. psittaci GPIC. These results suggest that the C. trachomatis IncA protein is involved in homotypic vesicle fusion and/or septation of the inclusion membrane that is believed to accompany bacterial cell division in C. psittaci . This proposal is corroborated by the expression of C. trachomatis and C. psittaci IncA in a yeast two-hybrid system to demonstrate C. trachomatis , but not C. psittaci , IncA interactions. Despite the inhibition of homotypic fusion of C. trachomatis inclusions, fusion of sphingomyelin-containing vesicles with the inclusion was not suppressed.     

Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release

Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl beta-cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4 degrees C but shifted to lipid rafts at 37 degrees C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.     

Low iron availability modulates the course of Chlamydia pneumoniae infection

Chlamydiae are obligate intracellular bacteria residing exclusively in host cell vesicles termed inclusions. We have investigated the effects of deferoxamine mesylate (DAM)-induced iron deficiency on the growth of Chlamydia pneumoniae and Chlamydia trachomatis serovar L2. In epithelial cells subjected to iron starvation and infected with either C. pneumoniae or C. trachomatis L2, small inclusions were formed, and the infectivity of chlamydial progeny was impaired. Moreover, for C. trachomatis L2, we observed a delay in homotypic fusion of inclusions. The inhibitory effects of DAM were reversed by adding exogenous iron-saturated transferrin, which restored the production of infectious chlamydiae. Electron microscopy examination of iron-deprived specimens revealed that the small inclusions contained reduced numbers of C. pneumoniae that were mostly reticulate bodies. We have previously reported specific accumulation of transferrin receptors (TfRs) around C. pneumoniae inclusions within cells grown under normal conditions. Using confocal and electron microscopy, we show here a remarkable increase in the amount of TfRs surrounding the inclusions in iron-starved cultures. It has been shown that iron is an essential factor in the growth and survival of C. trachomatis. Here, we postulate that, for C. pneumoniae also, iron is an indispensable element and that Chlamydia may use iron transport pathways of the host by attracting TfR to the phagosome.     

Uncoupling of the cholera toxin-G(M1) ganglioside receptor complex from endocytosis, retrograde Golgi trafficking, and downstream signal transduction by depletion of membrane cholesterol

To induce toxicity, cholera toxin (CT) must first bind ganglioside G(M1) at the plasma membrane, enter the cell by endocytosis, and then traffic retrograde into the endoplasmic reticulum. We recently proposed that G(M1) provides the sorting motif necessary for retrograde trafficking into the biosynthetic/secretory pathway of host cells, and that such trafficking depends on association with lipid rafts and lipid raft function. To test this idea, we examined whether CT action in human intestinal T84 cells depends on membrane cholesterol. Chelation of cholesterol with 2-hydroxypropyl beta-cyclodextrin or methyl beta-cyclodextrin reversibly inhibited CT-induced chloride secretion and prolonged the time required for CT to enter the cell and induce toxicity. These effects were specific to CT, as identical conditions did not alter the potency or toxicity of anthrax edema toxin that enters the cell by another mechanism. We found that endocytosis and trafficking of CT into the Golgi apparatus depended on membrane cholesterol. Cholesterol depletion also changed the density and specific protein content of CT-associated lipid raft fractions but did not entirely displace the CT-G(M1) complex from these lipid raft microdomains. Taken together these data imply that cholesterol may function to couple the CT-G(M1) complex with raft domains and with other membrane components of the lipid raft required for CT entry into the cell.     

Chlamydia trachomatis hijacks intra-Golgi COG complex-dependent vesicle trafficking pathway

Chlamydia spp. are obligate intracellular bacteria that replicate inside the host cell in a bacterial modified unique compartment called the inclusion. As other intracellular pathogens, chlamydiae exploit host membrane trafficking pathways to prevent lysosomal fusion and to acquire energy and nutrients essential for their survival and replication. The Conserved Oligomeric Golgi (COG) complex is a ubiquitously expressed membrane-associated protein complex that functions in a retrograde intra-Golgi trafficking through associations with coiled-coil tethers, SNAREs, Rabs and COPI proteins. Several COG complex-interacting proteins, including Rab1, Rab6, Rab14 and Syntaxin 6 are implicated in chlamydial development. In this study, we analysed the recruitment of the COG complex and GS15-positive COG complex-dependent vesicles to Chlamydia trachomatis inclusion and their participation in chlamydial growth. Immunofluorescent analysis revealed that both GFP-tagged and endogenous COG complex subunits associated with inclusions in a serovar-independent manner by 8 h post infection and were maintained throughout the entire developmental cycle. Golgi v-SNARE GS15 was associated with inclusions 24 h post infection, but was absent on the mid-cycle (8 h) inclusions, indicating that this Golgi SNARE is directed to inclusions after COG complex recruitment. Silencing of COG8 and GS15 by siRNA significantly decreased infectious yield of chlamydiae. Further, membranous structures likely derived from lysed bacteria were observed inside inclusions by electron microscopy in cells depleted of COG8 or GS15. Our results showed that C. trachomatis hijacks the COG complex to redirect the population of Golgi-derived retrograde vesicles to inclusions. These vesicles likely deliver nutrients that are required for bacterial development and replication.     

Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation

Caveolae are a membrane specialization used to internalize molecules by potocytosis. Caveolin, an integral membrane protein, is associated with the striated coat present on the cytoplasmic surface of the caveolae membrane. We now report that oxidation of caveolar cholesterol with cholesterol oxidase rapidly displaces the caveolin from the plasma membrane to intracellular vesicles that colocalize with Golgi apparatus markers. After the enzyme is removed from the medium, caveolin returns to caveolae. When untreated cells are gently homogenized, caveolin on the plasma membrane is accessible to both anti-caveolin IgG and trypsin. After cholesterol oxidase treatment, however, Golgi-associated caveolin is inaccessible to both of these molecules. Brefeldin A, which inhibits ER to Golgi trafficking, blocks the appearance of caveolin in the Golgi apparatus but does not prevent caveolin from leaving the plasma membrane. Indirect immunogold localization experiments show that in the presence of cholesterol oxidase caveolin leaves the plasma membrane and becomes associated with endoplasmic reticulum and Golgi compartments. Surprisingly, the loss of caveolin from the plasma membrane does not affect the number or morphology of the caveolae.     

Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity

In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to < 50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3.     

Cellular entry of lymphocytic choriomeningitis virus

In contrast to most enveloped viruses that enter the host cell via clathrin-dependent endocytosis, the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) enters cells via noncoated vesicles that deliver the virus to endosomes, where pH-dependent membrane fusion occurs. Here, we investigated the initial steps of LCMV infection. We found that the attachment of LCMV to its cellular receptor α-dystroglycan occurs rapidly and is not dependent on membrane cholesterol. However, subsequent virus internalization is sensitive to cholesterol depletion, indicating the involvement of a cholesterol-dependent pathway. We provide evidence that LCMV entry involves an endocytotic pathway that is independent of clathrin and caveolin and that does not require the GTPase dynamin. In addition, neither the structural integrity nor the dynamics of the actin cytoskeleton are required for infection. These findings indicate that the prototypic Old World arenavirus LCMV uses a mechanism of entry that is different from clathrin-mediated endocytosis, which is used by the New World arenavirus Junin virus, and pathways used by other enveloped viruses.     

Distribution and transport of cholesterol-rich membrane domains monitored by a membrane-impermeant fluorescent polyethylene glycol-derivatized cholesterol

Cholesterol-rich membrane domains function in various membrane events as diverse as signal transduction and membrane traffic. We studied the interaction of a fluorescein ester of polyethylene glycol-derivatized cholesterol (fPEG-Chol) with cholesterol-rich membranes both in cells and in model membranes. Unlike filipin and other cholesterol probes, this molecule could be applied as an aqueous dispersion to various samples. When added to live cells, fPEG-Chol distributed exclusively in the outer plasma membrane leaflet and was enriched in microdomains that dynamically clustered by the activation of receptor signaling. The surface-bound fPEG-Chol was slowly internalized via clathrin-independent pathway into endosomes together with lipid raft markers. Noteworthy, fPEG-Chol could be microinjected in the living cells in which we found Golgi apparatus as the sole major organelle to be labeled. PEG-Chol, thus, provides a novel, sensitive probe for unraveling the dynamics of cholesterol-rich microdomains in living cells.     

Cholesterol effectively blocks entry of flavivirus

Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2) are enveloped flaviviruses that enter cells through receptor-mediated endocytosis and low pH-triggered membrane fusion and then replicate in intracellular membrane structures. Lipid rafts, cholesterol-enriched lipid-ordered membrane domains, are platforms for a variety of cellular functions. In this study, we found that disruption of lipid raft formation by cholesterol depletion with methyl-beta-cyclodextrin or cholesterol chelation with filipin III reduces JEV and DEN-2 infection, mainly at the intracellular replication steps and, to a lesser extent, at viral entry. Using a membrane flotation assay, we found that several flaviviral nonstructural proteins are associated with detergent-resistant membrane structures, indicating that the replication complex of JEV and DEN-2 localizes to the membranes that possess the lipid raft property. Interestingly, we also found that addition of cholesterol readily blocks flaviviral infection, a result that contrasts with previous reports of other viruses, such as Sindbis virus, whose infectivity is enhanced by cholesterol. Cholesterol mainly affected the early step of the flavivirus life cycle, because the presence of cholesterol during viral adsorption greatly blocked JEV and DEN-2 infectivity. Flavirial entry, probably at fusion and RNA uncoating steps, was hindered by cholesterol. Our results thus suggest a stringent requirement for membrane components, especially with respect to the amount of cholesterol, in various steps of the flavivirus life cycle.     

Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.     

Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Summary Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.     

Lipid raft-dependent uptake, signalling and intracellular fate of Porphyromonas gingivalis in mouse macrophages

Lipid rafts are cholesterol-enriched microdomains involved in cellular trafficking and implicated as portals for certain pathogens. We sought to determine whether the oral pathogen Porphyromonas gingivalis enters macrophages via lipid rafts, and if so, to examine the impact of raft entry on its intracellular fate. Using J774A.1 mouse macrophages, we found that P. gingivalis colocalizes with lipid rafts in a cholesterol-dependent way. Depletion of cellular cholesterol using methyl-beta-cyclodextrin resulted in about 50% inhibition of P. gingivalis uptake, although this effect was reversed by cholesterol reconstitution. The intracellular survival of P. gingivalis was dramatically inhibited in cholesterol-depleted cells relative to untreated or cholesterol-reconstituted cells, even when infections were adjusted to allow equilibration of the initial intracellular bacterial load. P. gingivalis thus appeared to exploit raft-mediated uptake for promoting its survival. Consistent with this, lipid raft disruption enhanced the colocalization of internalized P. gingivalis with lysosomes. In contrast, raft disruption did not affect the expression of host receptors interacting with P. gingivalis, although it significantly inhibited signal transduction. In summary, P. gingivalis uses macrophage lipid rafts as signalling and entry platforms, which determine its intracellular fate to the pathogen's own advantage.     

Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions

Methyl-beta-cyclodextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [3H]-cholesterol. It was found that at 37 degrees C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 degrees C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 degrees C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.     

Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition

The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.     

Intra-Golgi protein transport depends on a cholesterol balance in the lipid membrane

Transport of proteins between intracellular membrane compartments is mediated by a protein machinery that regulates the budding and fusion processes of individual transport steps. Although the core proteins of both processes are defined at great detail, much less is known about the involvement of lipids. Here we report that changing the cellular balance of cholesterol resulted in changes of the morphology of the Golgi apparatus, accompanied by an inhibition of protein transport. By using a well characterized cell-free intra-Golgi transport assay, these observations were further investigated, and it was found that the transport reaction is sensitive to small changes in the cholesterol content of Golgi membranes. Addition as well as removal of cholesterol (10 +/- 6%) to Golgi membranes by use of methyl-beta-cyclodextrin specifically inhibited the intra-Golgi transport assay. Transport inhibition occurred at the fusion step. Modulation of the cholesterol content changed the lipid raft partitioning of phosphatidylcholine and heterotrimeric G proteins, but not of other (non) lipid raft proteins and lipids. We suggest that the cholesterol balance in Golgi membranes plays an essential role in intra-Golgi protein transport and needs to be carefully regulated to maintain the structural and functional organization of the Golgi apparatus.     

Orientation of glycoprotein galactosyltransferase and sialyltransferase enzymes in vesicles derived from rat liver Golgi apparatus

UDP-galactose: N-acetylglucosamine galactosyltransferase (GT) and CMP- sialic:desialylated transferrin sialyltransferse (ST) activities of rat liver Golgi apparatus are membrane-bound enzymes that can be released by treatment with Triton X-100. When protein substrates are used to assay these enzymes in freshly prepared Golgi vesicles, both activities are enhanced about eightfold by the addition of Triton X-100. When small molecular weight substrates are used, however, both activities are only enhanced about twofold by the addition of detergent. The enzymes remain inaccessible to large protein substrates even after freezing and storage of the Golgi preparation for 2 mo in liquid nitrogen. Accessibility to small molecular and weight substrates increases significantly after such storage. GT and ST activities in Golgi vesicles are not destroyed by treatment with trypsin, but are destroyed by this treatment if the vesicles are first disrupted with Triton X-100. Treatment of Golgi vesicles with low levels of filipin, a polyene antibiotic known to complex with cholesterol in biological membranes, also results in enhanced trypsin susceptibility of both glycosyltransferases. Maximum destruction of the glycosyltransferase activities by trypsin is obtained at filipin to total cholesterol weight ratios of approximately 1.6 or molar ratios of approximately 1. This level of filipin does not solubilize the enzymes but causes both puckering of Golgi membranes visible by electron microscopy and disruption of the Golgi vesicles as measured by release of serum albumin. When isolated Golgi apparatus is fixed with glutaraldehyde to maintain the three-dimensional orientation of cisternae and secretory vesicles, and then treated with filipin, cisternal membranes on both cis and trans faces of the apparatus as well as secretory granule membranes appear to be affected about equally. These results indicate that liver Golgi vesicles as isolated are largely oriented with GT and ST on the luminal side of the membranes, which corresponds to the cisternal compartment of the Golgi apparatus in the hepatocyte. Cholesterol is an integral part of the membrane of the Golgi apparatus and its distribution throughout the apparatus is similar to that of both transferases.     

Annexin A6-induced alterations in cholesterol transport and caveolin export from the Golgi complex

Annexin A6 (AnxA6) belongs to a family of Ca(2+)-dependent membrane-binding proteins and is involved in the regulation of endocytic and exocytic pathways. We previously demonstrated that AnxA6 regulates receptor-mediated endocytosis and lysosomal targeting of low-density lipoproteins and translocates to cholesterol-enriched late endosomes (LE). As cholesterol modulates the membrane binding and the cellular location of AnxA6, but also affects the intracellular distribution of caveolin, we investigated the localization and trafficking of caveolin in AnxA6-expressing cells. Here, we show that cells expressing high levels of AnxA6 are characterized by an accumulation of caveolin-1 (cav-1) in the Golgi complex. This is associated with a sequestration of cholesterol in the LE and lower levels of cholesterol in the Golgi and the plasma membrane, both likely contributing to retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. Further strengthening these findings, knock down of AnxA6 and the ectopic expression of the Niemann-Pick C1 protein in AnxA6-overexpressing cells restore the cellular distribution of cav-1 and cholesterol, respectively. In summary, this study demonstrates that elevated expression levels of AnxA6 perturb the intracellular distribution of cholesterol, which indirectly inhibits the exit of caveolin from the Golgi complex.