Lipid remodeling of GPI-anchored proteins and its function

Many proteins are attached to the cell surface via a conserved post-translational modification, the glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins are functionally diverse, but one of their most striking features is their association with lipid microdomains, which consist mainly of sphingolipids and sterols. GPI-anchored proteins modulate various biological functions when they are incorporated into these specialized domains. The biosynthesis of GPI and its attachment to proteins occurs in the endoplasmic reticulum. The lipid moieties of GPI-anchored proteins are further modified during their transport to the cell surface, and these remodeling processes are essential for the association of proteins with lipid microdomains. Recently, several genes required for GPI lipid remodeling have been identified in yeast and mammalian cells. In this review, we describe the pathways for lipid remodeling of GPI-anchored proteins in yeast and mammalian cells, and discuss how lipid remodeling affects the association of GPI-anchored proteins with microdomains in cellular events.     

Possible roles of glycosphingolipids in lipid rafts

Recent studies have suggested that glycosphingolipid (GSL)-cholesterol microdomains in cell membranes may function as platforms for the attachment of lipid-modified proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins and src-family tyrosine kinases. The microdomains are proposed to be involved in membrane trafficking of GPI-anchored proteins and in signal transduction via src-family kinases. Here, the possible roles of GSLs in the physical properties of these microdomains, as well as in membrane trafficking and signal transduction, are discussed. Sphingolipid depletion inhibits the intracellular transport of GPI-anchored proteins in biosynthetic traffic and endocytosis via GPI-anchored proteins. Antibodies against GSLs as well as GPI-anchored proteins co-precipitate src-family kinases. Antibody-mediated cross-linking of GSLs, as well as that of GPI-anchored proteins, induces a transient increase in the tyrosine phosphorylation of several substrates. Thus, GSLs have important roles in lipid rafts.     

Roles for L o microdomains and ESCRT in ER stress-induced lipid droplet microautophagy in budding yeast

Microlipophagy (µLP), degradation of lipid droplets (LDs) by microautophagy, occurs by autophagosome-independent direct uptake of LDs at lysosomes/vacuoles in response to nutrient limitations and ER stressors in Saccharomyces cerevisiae. In nutrient-limited yeast, liquid-ordered (L_o) microdomains, sterol-rich raftlike regions in vacuolar membranes, are sites of membrane invagination during LD uptake. The endosome sorting complex required for transport (ESCRT) is required for sterol transport during L_o formation under these conditions. However, ESCRT has been implicated in mediating membrane invagination during µLP induced by ER stressors or the diauxic shift from glycolysis- to respiration-driven growth. Here we report that ER stress induced by lipid imbalance and other stressors induces L_o microdomain formation. This process is ESCRT independent and dependent on Niemann-Pick type C sterol transfer proteins. Inhibition of ESCRT or L_o microdomain formation partially inhibits lipid imbalance-induced µLP, while inhibition of both blocks this µLP. Finally, although the ER stressors dithiothreitol or tunicamycin induce L_o microdomains, µLP in response to these stressors is ESCRT dependent and L_o microdomain independent. Our findings reveal that L_o microdomain formation is a yeast stress response, and stress-induced L_o microdomain formation occurs by stressor-specific mechanisms. Moreover, ESCRT and L_o microdomains play functionally distinct roles in LD uptake during stress-induced µLP.     

Glycosylphosphatidylinositol-anchored proteins: structure, function, and cleavage by phosphatidylinositol-specific phospholipase C.

A wide variety of proteins are tethered by a glycosylphosphatidylinositol (GPI) anchor to the extracellular face of eukaryotic plasma membranes, where they are involved in a number of functions ranging from enzymatic catalysis to adhesion. The exact function of the GPI anchor has been the subject of much speculation. It appears to act as an intracellular signal targeting proteins to the apical surface in polarized cells. GPI-anchored proteins are sorted into sphingolipid- and cholesterol-rich microdomains, known as lipid rafts, before transport to the membrane surface. Their localization in raft microdomains may explain the involvement of this class of proteins in signal transduction processes. Substantial evidence suggests that GPI-anchored proteins may interact closely with the bilayer surface, so that their functions may be modulated by the biophysical properties of the membrane. The presence of the anchor appears to impose conformational restraints, and its removal may alter the catalytic properties and structure of a GPI-anchored protein. Release of GPI-anchored proteins from the cell surface by specific phospholipases may play a key role in regulation of their surface expression and functional properties. Reconstitution of GPI-anchored proteins into bilayers of defined phospholipids provides a powerful tool with which to explore the interactions of these proteins with the membrane and investigate how bilayer properties modulate their structure, function, and cleavage by phospholipases.     

Association of a GPI-anchored protein with detergent-resistant membranes facilitates its trafficking through the early secretory pathway

Membrane microdomains are implicated in the trafficking and sorting of several membrane proteins. In particular GPI-anchored proteins cluster into Triton X-100 resistant, cholesterol- and sphingolipid-rich membrane microdomains and are sorted to the apical membrane. A growing body of evidence has pointed to the existence of other types of microdomains that are insoluble in detergents, such as Lubrol WX and Tween-20. Here, we report on the role of detergent-resistant membranes formed at early stages in the biosynthesis of membrane dipeptidase (MDP), a GPI-anchored protein, on its trafficking and sorting. Pulse-chase experiments revealed a retarded maturation rate of the GPI-anchor deficient mutant (MDPΔGPI) as compared to the wild type protein (wtMDP). However, Golgi to cell surface delivery rate did not show a significant difference between the two variants. On the other hand, early biosynthetic forms of wtMDP were partially insoluble in Tween-20, while MDPΔGPI was completely soluble. The lack of association of MDPΔGPI with detergent-resistant membranes prior to maturation in the Golgi and the reduction in its trafficking rate strongly suggest the existence of an early trafficking control mechanisms for membrane proteins operating at a level between the endoplasmic reticulum and the cis-Golgi.     

Protein sorting upon exit from the endoplasmic reticulum

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.     

Functional roles of glycosphingolipids in signal transduction via lipid rafts

The formation of glycosphingolipid (GSL)-cholesterol microdomains in cell membranes has been proposed to function as platforms for the attachment of lipid-modified proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins and src-family tyrosine kinases. The microdomains are postulated to be involved in GPI-anchored protein signaling via src-family kinase. Here, the functional roles of GSLs in signal transduction mediated by the microdomains are discussed. Antibodies against GSLs co-precipitate GPI-anchored proteins, src-family kinases and several components of the microdomains. Antibody-mediated crosslinking of GSLs, as well as that of GPI-anchored proteins, induces a rapid activation of src-family kinases and a transient increase in the tyrosine phosphorylation of several substrates. Enzymatic degradation of GSLs reduces the activation of src-family kinase and tyrosine phosphorylation by antibody-mediated crosslinking of GPI-anchored protein. Furthermore, GSLs can also modulate signal transduction of immunoreceptors and growth factor receptors in the microdomains. Thus, GSLs have important roles in signal transduction mediated by the microdomains.     

Functional roles of glycosphingolipids in signal transduction via lipid rafts

The formation of glycosphingolipid (GSL)-cholesterol microdomains in cell membranes has been proposed to function as platforms for the attachment of lipid-modified proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins and src-family tyrosine kinases. The microdomains are postulated to be involved in GPI-anchored protein signaling via src-family kinase. Here, the functional roles of GSLs in signal transduction mediated by the microdomains are discussed. Antibodies against GSLs co-precipitate GPI-anchored proteins, src-family kinases and several components of the microdomains. Antibody-mediated crosslinking of GSLs, as well as that of GPI-anchored proteins, induces a rapid activation of src-family kinases and a transient increase in the tyrosine phosphorylation of several substrates. Enzymatic degradation of GSLs reduces the activation of src-family kinase and tyrosine phosphorylation by antibody-mediated crosslinking of GPI-anchored protein. Furthermore, GSLs can also modulate signal transduction of immunoreceptors and growth factor receptors in the microdomains. Thus, GSLs have important roles in signal transduction mediated by the microdomains.     

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.     

Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.

We show that a protein with a glycosylphosphatidyl inositol (GPI) anchor can be recovered from lysates of epithelial cells in a low density, detergent-insoluble form. Under these conditions, the protein is associated with detergent-resistant sheets and vesicles that contain other GPI-anchored proteins and are enriched in glycosphingolipids, but do not contain a basolateral marker protein. The protein is recovered in this complex only after it has been transported to the Golgi complex, suggesting that protein-sphingolipid microdomains form in the Golgi apparatus and plasma membrane and supporting the model proposed by Simons and colleagues for sorting of certain membrane proteins to the apical surface after intracellular association with glycosphingolipids.     

GPI-anchor remodeling: potential functions of GPI-anchors in intracellular trafficking and membrane dynamics

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved post-translational modification in eukaryotes. GPI is synthesized and transferred to proteins in the endoplasmic reticulum. GPI-anchored proteins are then transported from the endoplasmic reticulum to the plasma membrane through the Golgi apparatus. GPI-anchor functions as a sorting signal for transport of GPI-anchored proteins in the secretory and endocytic pathways. After GPI attachment to proteins, the structure of the GPI-anchor is remodeled, which regulates the trafficking and localization of GPI-anchored proteins. Recently, genes required for GPI remodeling were identified in yeast and mammalian cells. Here, we describe the structural remodeling and function of GPI-anchors, and discuss how GPI-anchors regulate protein sorting, trafficking, and dynamics. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes

"Lipid rafts" enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.     

Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway

Epithelial cell polarity depends on mechanisms for targeting proteins to different plasma membrane domains. Here, we dissect the pathway for apical delivery of several raft-associated, glycosyl phosphatidylinositol (GPI)-anchored proteins in polarized MDCK cells using live-cell imaging and selective inhibition of apical or basolateral exocytosis. Rather than trafficking directly from the trans-Golgi network (TGN) to the apical plasma membrane as previously thought, the GPI-anchored proteins followed an indirect, transcytotic route. They first exited the TGN in membrane-bound carriers that also contained basolateral cargo, although the two cargoes were laterally segregated. The carriers were then targeted to and fused with a zone of lateral plasma membrane adjacent to tight junctions that is known to contain the exocyst. Thereafter, the GPI-anchored proteins, but not basolateral cargo, were rapidly internalized, together with endocytic tracer, into clathrin-free transport intermediates that transcytosed to the apical plasma membrane. Thus, apical sorting of these GPI-anchored proteins occurs at the plasma membrane, rather than at the TGN.     

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.     

Identification of signals and mechanisms of sorting of plasma membrane proteins in intestinal epithelial cells]

In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.     

The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.     

Detergent-insoluble GPI-anchored proteins are apically sorted in fischer rat thyroid cells, but interference with cholesterol or sphingolipids differentially affects detergent insolubility and apical sorting

In contrast to Madin-Darby canine kidney cells, Fischer rat thyroid cells deliver the majority of endogenous glycosylphosphatidyl inositol (GPI)-anchored proteins to the basolateral surface. However, we report here that the GPI proteins Placental Alkaline Phosphatase (PLAP) and Neurotrophin Receptor-Placental Alkaline Phosphatase (NTR-PLAP) are apically localized in transfected Fischer rat thyroid cells. In agreement with the "raft hypothesis," which postulates the incorporation of GPI proteins into glycosphingolipids and cholesterol-enriched rafts, we found that both of these proteins were insoluble in Triton X-100 and floated into the lighter fractions of sucrose density gradients. However, disruption of lipid rafts by removal of cholesterol did not cause surface missorting of PLAP and NTR-PLAP, and the altered surface sorting of these proteins after Fumonisin B1 treatment did not correlate with reduced levels in Triton X-100 -insoluble fractions. Furthermore, in contrast to the GPI-anchored forms of both of these proteins, the secretory and transmembrane forms (in the absence of a basolateral cytoplasmic signal) were sorted to the apical surface without association with lipid microdomains. Together, these data demonstrate that the GPI anchor is required to mediate raft association but is not sufficient to determine apical sorting. They also suggest that signals present in the ectodomain of the proteins play a major role and that lipid rafts may facilitate the recognition of these signals in the trans-Golgi network, even though they are not required for apical sorting.     

Inhibition of sphingolipid synthesis: effects on glycosphingolipid-GPI-anchored protein microdomains

The idea that the transport and sorting of glycosylphosphatidylinositol (GPI)-anchored proteins depends on their interaction with glycosphingolipids was first proposed five or six years ago. Until recently, only circumstantial evidence was available to support this suggestion. During the past year, compelling support for this hypothesis has been provided by observations that inhibition of sphingolipid synthesis reduces the rate of transport of GPI-anchored proteins in yeast, and abolishes the polarized sorting of a GPI-anchored protein in epithelia.     

Differential endocytic functions of Trypanosoma brucei Rab5 isoforms reveal a glycosylphosphatidylinositol-specific endosomal pathway.

We demonstrate the presence of a glycosylphosphatidylinositol (GPI) anchor-specific endosomal pathway in the protozoan pathogen Trypanosoma brucei. In higher eukaryotes evidence indicates that GPI-anchored proteins are transported in both the endocytic and exocytic systems by mechanisms involving sequestration into specific membrane microdomains and consequently sorting into distinct compartments. This is potentially extremely important in trypanosomatids as the GPI anchor is the predominant mechanism for membrane attachment of surface macromolecules, including the variant surface glycoprotein (VSG). A highly complex developmentally regulated endocytic network, vital for nutrient uptake and evasion of the immune response, exists in T. brucei. In common with mammalian cells an early endosomal compartment is defined by Rab5 small GTPases, which control transport processes through the endosomal system. We investigate the function of two trypanosome Rab5 homologues. TbRAB5A and TbRAB5B, which colocalize in the procyclic stage, are distinct in the bloodstream form of the parasite. TbRAB5A endosomes contain VSG and transferrin, endocytosed by the T. brucei GPI-anchored transferrin receptor, whereas TbRAB5B endosomes contain the transmembrane protein ISG(100) but neither VSG nor transferrin. These findings indicate the presence of trypanosome endosomal pathways trafficking proteins through specific routes depending on the mode of membrane attachment. Ectopic expression of mutant TbRAB5A or -5B indicates that TbRAB5A plays a role in LDL endocytosis, whereas TbRAB5B does not, but both have a role in fluid phase endocytosis. Hence TbRAB5A and TbRAB5B have distinct functions in the endosomal system of T. brucei. A developmentally regulated GPI-specific endosomal pathway in the bloodstream form suggests that specialized transport of GPI-anchored proteins is required for survival in the mammalian host.     

The presence of an ER exit signal determines the protein sorting upon ER exit in yeast

In yeast, there are at least two vesicle populations upon ER (endoplasmic reticulum) exit, one containing Gap1p (general aminoacid permease) and a glycosylated alpha-factor, gpalphaF (glycosylated proalpha-factor), and the other containing GPI (glycosylphosphatidylinositol)-anchored proteins, Gas1p (glycophospholipid-anchored surface protein) and Yps1p. We attempted to identify sorting determinants for this protein sorting event in the ER. We found that mutant Gas1 proteins that lack a GPI anchor and/or S/T region (serine- and threonine-rich region), two common characteristic features conserved among yeast GPI-anchored proteins, were still sorted away from Gap1p-containing vesicles. Furthermore, a mutant glycosylated alpha-factor, gpalphaGPI, which contains both the GPI anchor and S/T region from Gas1p, still entered Gap1p-containing vesicles, demonstrating that these conserved characteristics do not prevent proteins from entering Gap1p-containing vesicles. gpalphaF showed severely reduced budding efficiency in the absence of its ER exit receptor Erv29p, and this residual budding product no longer entered Gap1p-containing vesicles. These results suggest that the interaction of gpalphaF with Erv29p is essential for sorting into Gap1p-containing vesicles. We compared the detergent solubility of Gas1p and the gpalphaGPI in the ER with that in ER-derived vesicles. Both GPI-anchored proteins similarly partitioned into the DRM (detergent-resistant membrane) in the ER. Based on the fact that they entered different ER-derived vesicles, we conclude that DRM partitioning of GPI-anchored proteins is not the dominant determinant of protein sorting upon ER exit. Interestingly, upon incorporation into the ER-derived vesicles, gpalphaGPI was no longer detergent-insoluble, in contrast with the persistent detergent insolubility of Gas1p in the ER-derived vesicles. We present different explanations for the different behaviours of GPI-anchored proteins in distinct ER-derived vesicle populations.