Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins

An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein-protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.     

A Highlights from MBoC Selection: Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation

Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin–Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.     

GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin-Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.     

Oligomerization is a specific requirement for apical sorting of glycosyl-phosphatidylinositol-anchored proteins but not for non-raft-associated apical proteins

Protein apical sorting in polarized epithelial cells is mediated by two different mechanisms, raft dependent and raft independent. In Madin-Darby canine kidney (MDCK) cells, an essential step for apical sorting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) is their coalescence into high-molecular-weight (HMW) oligomers. Here we show that this mechanism is also functional in Fischer rat thyroid cells, which possess a different sorting phenotype compared with MDCK cells. We demonstrate that, as in MDCK cells, both apical and basolateral GPI-APs associate with detergent-resistant microdomains, but that only the apical proteins are able to oligomerize into HMW complexes during their passage through the medial Golgi. We also show that oligomerization is a specific requirement for apical sorting of GPI-APs and is not used by transmembrane, non-raft-associated apical proteins.     

N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells

Sorting of glycosylphosphatidyl-inositol--anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin-Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.     

Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells

We have previously reported that 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAc alpha-O-bn), an inhibitor of glycosylation, perturbed apical biosynthetic trafficking in polarized HT-29 cells suggesting an involvement of a lectin-based mechanism. Here, we have identified galectin-4 as one of the major components of detergent-resistant membranes (DRMs) isolated from HT-29 5M12 cells. Galectin-4 was also found in post-Golgi carrier vesicles. The functional role of galectin-4 in polarized trafficking in HT-29 5M12 cells was studied by using a retrovirus-mediated RNA interference. In galectin-4-depleted HT-29 5M12 cells apical membrane markers accumulated intracellularly. In contrast, basolateral membrane markers were not affected. Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins. Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4. Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.     

N- and O-glycans are not directly involved in the oligomerization and apical sorting of GPI proteins

Oligomerization of glycosyl-phosphatidylinositol-anchored proteins (GPI-APs) into high molecular weight complexes is an essential step for their apical sorting in polarized epithelial cells. However, the mechanism by which apical GPI-APs oligomerize is still unclear. To investigate the possible role of N- and O-glycosylation, we have analysed the behaviour of two glycosylated GPI-anchored apical proteins, p75GPI and placental alkaline phosphatase (PLAP), and their glycosylation mutants. We found that both the N- and O-glycosylation mutants are apically sorted, associate to detergent-resistant microdomains and are able to oligomerize, like the wild-type proteins, suggesting that glycosylation does not have a direct role in GPI-AP oligomerization and apical sorting. Interestingly, when cells are depleted of cholesterol and treated with tunicamycin, treatments that by themselves do not affect PLAP sorting, PLAP is not able to oligomerize and is missorted to the basolateral surface, thus supporting an indirect role of N-glycosylation, possibly mediated by a raft-associated glycosylated interactor.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 degrees C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Cytoskeleton-dependent Membrane Domain Segregation during Neutrophil Polarization

On treatment with chemoattractant, the neutrophil plasma membrane becomes organized into detergent-resistant membrane domains (DRMs), the distribution of which is intimately correlated with cell polarization. Plasma membrane at the front of polarized cells is susceptible to extraction by cold Triton X-100, whereas membrane at the rear is resistant to extraction. After cold Triton X-100 extraction, DRM components, including the transmembrane proteins CD44 and CD43, the GPI-linked CD16, and the lipid analog, DiIC(16), are retained within uropods and cell bodies. Furthermore, CD44 and CD43 interact concomitantly with DRMs and with the F-actin cytoskeleton, suggesting a mechanism for the formation and stabilization of DRMs. By tracking the distribution of DRMs during polarization, we demonstrate that DRMs progress from a uniform distribution in unstimulated cells to small, discrete patches immediately after activation. Within 1 min, DRMs form a large cap comprising the cell body and uropod. This process is dependent on myosin in that an inhibitor of myosin light chain kinase can arrest DRM reorganization and cell polarization. Colabeling DRMs and F-actin revealed a correlation between DRM distribution and F-actin remodeling, suggesting that plasma membrane organization may orient signaling events that control cytoskeletal rearrangements and, consequently, cell polarity.     

Increased association with detergent-resistant membranes/lipid rafts of apically targeted mutants of the interleukin-6 receptor gp80

Interleukin (IL)-6 is an important cytokine in inflammatory processes, differentiation and growth. The IL-6 receptor complex comprises the specific IL-6 receptor (gp80) and two molecules of the signal tranducing component gp130 which transduces the signal into the nucleus via the Jak-STAT pathway. Both, gp80 and gp130 are sorted preferentially to the basolateral membrane of polarised Madin-Darby canine kidney (MDCK) cells. Previously, we have shown that gp130 partially localises to detergent-resistant membranes (DRMs)/lipid rafts and that lipid raft integrity is crucial for signalling to occur. Here we now demonstrate that wild-type gp80 is associated with DRMs only to a minor extent. However, gp80 mutants which lack parts of the cytoplasmic domain and therefore are more apically expressed than the wild type show an increased affinity for the liquid-ordered membrane domain. Studies with non-polarised MDCK cells suggest that the lipid raft association of the different mutants of gp80 precedes the establishment of cell polarity. Our findings suggest that lipid rafts play a role in the sorting of apically targeted gp80.     

Detergent insoluble microdomains are not involved in transcytosis of polymeric Ig receptor in FRT and MDCK cells

In polarized epithelial cells, sorting of proteins and lipids to the apical or basolateral domain of the plasma membrane can occur via direct or indirect (transcytotic) pathways from the trans Golgi network (TGN). The 'rafts' hypothesis postulates that the key event for direct apical sorting of some transmembrane proteins and the majority of GPI-anchored proteins depends on their association with glycosphingolipid and cholesterol enriched microdomains (rafts). However, the mechanism of indirect sorting to the apical membrane is not clear. The polyimmunoglobulin receptor (pIgR) is one of the best studied proteins that follow the transcytotic pathway. It is normally delivered from the TGN to the basolateral surface of polarized Madin–Darby Canine Kidney (MDCK) cells from where it transports dIgA or dIgM to the apical surface. We have studied the intracellular trafficking of pIgR in Fischer rat thyroid cells (FRT), and have investigated the sorting machinery involved in transcytosis of this receptor in both FRT and MDCK cells. We found that, in contrast with MDCK cells, a significant amount (∼30%) of pIgR reaches the apical surface by a direct pathway. Furthermore, in both cell lines it does not associate with Triton X-100-insoluble microdomains, suggesting that at least in these cells 'rafts' are not involved in basolateral to apical transcytosis.     

Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells

Polarity is a central feature of eukaryotic cells and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, PtdIns(3,4,5)P3 is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP fused to the PtdIns(3,4,5)P3-binding pleckstrin-homology domain of Akt (GFP-PH-Akt), a fusion protein that indicates the location of PtdIns(3,4,5)P3. Here, we ectopically inserted exogenous PtdIns(3,4,5)P3 into the apical plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells. Within 5 min many cells formed protrusions that extended above the apical surface. These protrusions contained basolateral plasma membrane proteins and excluded apical proteins, indicating that their plasma membrane was transformed from apical to basolateral. Addition of PtdIns(3,4,5)P3 to the basolateral surface of MDCK cells grown as cysts caused basolateral protrusions. MDCK cells grown in the presence of a phosphatidylinositol 3-kinase inhibitor had abnormally short lateral surfaces, indicating that PtdIns(3,4,5)P3 regulates the formation of the basolateral surface.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 °C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.     

Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root development. We here present a general mass spectrometry-based proteomic "shave-and-conquer" strategy that specifically targets GPI-APs. Using a combination of biochemical methods, mass spectrometry, and computational sequence analysis we identified six GPI-APs in a Homo sapiens lipid raft-enriched fraction and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date.     

Distinct Lipid Rafts in Subdomains from Human Placental Apical Syncytiotrophoblast Membranes

We report on the characteristics of raft domains in the apical membrane from human placental syncytiotrophoblast (hSTB), an epithelium responsible for maternal-fetal exchange. Previously, we described two isolated fractions of the hSTB apical membrane: a classical microvillous membrane (MVM) and a light microvillous membrane (LMVM). Detergent-resistant microdomains (DRMs) from MVM and LMVM were prepared with Triton X-100 followed by flotation in a sucrose gradient and tested by Western and dot blot with raft markers (placental alkaline phosphatase, lipid ganglioside, annexin 2) and transferrin receptor as a nonraft marker. DRMs from both fractions showed a consistent peak for these markers, except that the DRMs from MVM had no annexin 2 mark. Cholesterol depletion modified the segregation in both groups of DRMs. Our results show two distinguishable lipid raft subsets from MVM and LMVM. Additionally, we found significant differences between MVM and LMVM in cholesterol content and in expression of cytoskeletal proteins. MVM is enriched in ezrin and beta-actin; in contrast, cholesterol and cytokeratin-7 are more abundant in LMVM. These differences may explain the distinct properties of the lipid raft subtypes.     

SNARE protein trafficking in polarized MDCK cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains. This polarity is generated and maintained by the continuous sorting of apical and basolateral components in the secretory and endocytic pathways. Soluble N-ethyl maleimide-sensitive factor attachment protein receptors (SNARE) proteins of vesicle-associated membrane protein (VAMP) and syntaxin families have been suggested to play a role in the biosynthetic transport to the apical and basolateral plasma membranes of polarized cells, where they likely mediate membrane fusion. To investigate the involvement of SNARE proteins in membrane trafficking to the apical and basolateral plasma membrane in the endocytic pathway we have monitored the recycling of various VAMP and syntaxin molecules between intracellular compartments and the two plasma membrane domains in Madin–Darby canine kidney (MDCK) cells. Here we show that VAMP8/endobrevin cycles through the apical but not through the basolateral plasma membrane. Furthermore, we found that VAMP8 localizes to apical endosomal membranes in nephric tubule epithelium and in MDCK cells. This asymmetry in localization and cycling behavior suggests that VAMP8/endobrevin may play a role in apical endosomal trafficking in polarized epithelium cells.     

Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.     

Protein-lipid interactions in antipodal plasma membranes of rat colonocytes

The isolation of apical membranes from rat proximal colonic epithelial cells is described. Differential centrifugation yielded a ‘crude’ membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 20–28-fold over homogenate in alkaline phosphatase and cysteine-sensitive alkaline phosphatase specific activities. Lipid-protein interactions and lipid dynamics examined in apical and basolateral membranes prepared from colonocytes demonstrated: (1) apical membrane, as assessed by steady-state fluorescence polarization studies have a low lipid fluidity; (2) colonic basolateral membranes possess a greater lipid fluidity than apical membranes; (3) compositional differences in these antipodal membranes appear to explain these differences in lipid fluidity; (4) fluorescence polarization studies using diphenylhexatriene detect a thermotropic transition at 21–23°C in apical membranes and liposomes prepared from lipid extracts of these membranes; (5) alkaline phosphatase and l-cysteine-sensitive alkaline phosphatase activities appear to be functionally dependent on the physical state of the apical membrane's lipid.