Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface

In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15°C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.

Segregation of Heterotrimeric G Proteins in Cell Surface Microdomains: Gq Binds Caveolin to Concentrate in Caveolae, whereas Gi and Gs Target Lipid Rafts by Default

Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinases may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (Gi, Gq, Gs, and Gβγ) target discrete cell surface microdomains. Gq specifically concentrates in caveolae, whereas Gi and Gs concentrate much more in lipid rafts marked by GPI-anchored proteins (5′ nucleotidase and folate receptor). Gq, apparently without Gβγ subunits, stably associates with plasmalemmal and cytosolic caveolin. Gi and Gs interact with Gβγ subunits but not caveolin. Gi and Gs, unlike Gq, readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize Gq preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, Gq, Gi, and Gs all concentrate in lipid rafts as a complex with Gβγ. Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the Gq-caveolin complex without Gβγ for trafficking, signaling, and mechanotransduction are discussed.

SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts (≈25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.

VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells

Apical proteins are sorted and delivered from the trans-Golgi network to the plasma membrane by a mechanism involving sphingolipid–cholesterol rafts. In this paper, we report the effects of changing the levels of VIP17/MAL, a tetraspan membrane protein localized to post-Golgi transport containers and the apical cell surface in MDCK cells. Overexpression of VIP17/MAL disturbed the morphology of the MDCK cell layers by increasing apical delivery and seemingly expanding the apical cell surface domains. On the other hand, expression of antisense RNA directed against VIP17/MAL caused accumulation in the Golgi and/or impaired apical transport of different apical protein markers, i.e., influenza virus hemagglutinin, the secretory protein clusterin (gp80), the transmembrane protein gp114, and a glycosylphosphatidylinositol-anchored protein. However, antisense RNA expression did not affect the distribution of E-cadherin to the basolateral surface. Because VIP17/MAL associates with sphingolipid–cholesterol rafts, these data provide functional evidence that this protein is involved in apical transport and might be a component of the machinery clustering lipid rafts with apical cargo to form apical transport carriers.

Raft association of SNAP receptors acting in apical trafficking in Madin–Darby canine kidney cells

We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin– Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.

Trafficking of spontaneously endocytosed MHC proteins

Class I MHC protein primarily presents endogenous antigen but also may present exogenous antigen. Here, we investigated the intracellular pathway of spontaneously internalized class I MHC protein by confocal microscopy. β2-microglobulin (β2m), labeled with a single fluorophore, was exchanged at the surface of B cell transfectants to specifically mark cell surface and endocytosed class I MHC protein. Intracellular β2m colocalized with fluorophore-conjugated transferrin, implying that class I MHC protein endocytosed into early endosomes. These endosomes containing fluorescent β2m were found close to or within the Golgi apparatus, marked by fluorescent ceramide. Even after 24 hr of incubation, very little fluorescent β2m was found in intracellular organelles stained by DiOC6, marking the endoplasmic reticulum, or fluorophore-conjugated low density lipoprotein, marking late endosomes and lysosomes. Fluorophore-conjugated superantigens (staphylococcal enterotoxin A and B), presumed to enter cells bound to class II MHC protein, also were found to endocytose into β2m-containing early endosomes. Staining with mAb and use of transfectants expressing MHC protein attached to green fluorescent protein confirmed the presence of intracellular compartments rich in both class I and II MHC protein and demonstrated that class I and II MHC protein also colocalize in discrete microdomains at the cell surface. These cell surface microdomains also contained transferrin receptor and often were juxtaposed to cholesterol-rich lipid rafts. Thus, class I and II MHC protein meet in microdomains of the plasma membrane and endocytose into early endosomes, where both may acquire and present exogenous antigen.

Apical sorting of a voltage- and Ca2+-activated K+ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

The voltage- and Ca2+-activated K+ (KV,Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel KV,Ca α-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., KV,Ca β-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.

Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling

The high affinity receptor for IgE, FcɛRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcɛRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcɛRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcɛRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcɛRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcɛRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcɛRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.

Epstein–Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: Protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors

Epstein–Barr virus encodes integral membrane proteins LMP1 and LMP2A in transformed lymphoblastoid cell lines. We now find that LMP1 associates with the cell cytoskeleton through a tumor necrosis factor receptor-associated factor-interacting domain, most likely mediated by tumor necrosis factor receptor-associated factor 3. LMP1 is palmitoylated, and the transmembrane domains associate with lipid rafts. Mutation of LMP1 cysteine-78 abrogates palmitoylation but does not affect raft association or NF-κB or c-Jun N-terminal kinase activation. LMP2A also associates with rafts and is palmitoylated but does not associate with the cell cytoskeleton. The associations of LMP1 and LMP2A with rafts and of LMP1 with the cell cytoskeleton are likely to effect interactions with cell proteins involved in shape, motility, signal transduction, growth, and survival.

An Intact Dilysine-like Motif in the Carboxyl Terminus of MAL Is Required for Normal Apical Transport of the Influenza Virus Hemagglutinin Cargo Protein in Epithelial Madin-Darby Canine Kidney Cells

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine −3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid–cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4°C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid–cholesterol microdomains in living cells.