Glycosylphosphatidylinositols: biosynthesis and intracellular transport

Glycoconjugate Biosynthesis

Glycosylphosphatidylinositols: biosynthesis and intracellular transport A. K. Menon*', N. A. Baumann*, W. van't Hoft and J. Vidugiriene*$ *Department of Biochemistry, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, U.S.A., tCell Biology and Genetics Program, Sloan-Kettering Institute, I275 York Avenue, New York, N Y I002 I , U.S.A., and *Department of Biochemistry and Biophysics, Vilnius University, Ciurlionio 2 I , Vilnius, Lithuania

Glycosylphosphatidylinositols(GPIs) are ubiquitous in eukaryotes. These structures, defined by elements of the sequence ethanolamine-P04Manor1-2Manor 1-6Manor 1-4GlcNa 1-6myo-inositol-PO4-lipid, exist covalently linked to selected proteins or as free glycolipids representing biosynthetic intermediates. GPI-linked proteins are confined to the exoplasmic leaflet of cellular membranes and are typically displayed at the cell surface. 'Free' GPIs display a wider subcellular Abbreviations used: ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol; MAM, mitochondrionassociated membrane; MDCK, Madin-Darby canine kidney epithelial cells; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PLD, phospholipase D; PS, phosphatidylserine; VSV, vesicular stomatitis virus. 'To whom correspondence should be addressed.

distribution. The purpose of this article is to discuss aspects of the biosynthesis and intracellular transport of free GPIs (henceforth referred to simply as GPIs), focusing mainly on new (unpublished) experimental observations obtained from analyses of mammalian cells.

Biosynthesis GPI biosynthesis (Figure 1, reviewed in [l-61) is initiated in the endoplasmic reticulum (ER) by the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI) to yield GlcNAc-PI. This reaction requires the participation of at least three gene products (designated A, C and H). In the second step of the biosynthetic path(defective in to mutant complementation class J), GlcNAc-PI is de-Nacetylated to yield GlcN-PI. In mammalian cells and yeast (but not in trypanosomes), the inositol

Model for the compartmentation of GPI biosynthesis in the ER and associated membranes The model is explained in detail in the text Modification of the GPI glycan with phosphoethanolamine groups (which could occur in region B) is not shown The letters K, A. C. H, J, E, B and F positioned at various steps of the biosynthetic scheme denote the complementation class of mammalian cell mutants defective in a particular biosynthetic step

n

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t Region A

Region B

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residue in GlcN-PI is esterified with a fatty acid to yield GlcN-acylPI. GlcN-acylPI (or GlcN-PI in trypanosomes) is elaborated via the transfer of at least three mannose residues (from dolicholP-Man), and one or more phosphoethanolamine groups linked to the mannose residues [the phosphoethanolamine residue linked to the third mannose from the reducing terminus of the glycan is derived from phosphatidylethanolamine (PE)]. Mannose transfer is blocked in the class E (defective in dolichol-P-Man synthesis) and class B (lacking the third GPI mannosyltransferase) mutants, and phosphoethanolamine transfer to the third mannose is defective in the class F mutant. Side-chain modifications of the core GPI structure may occur during assembly or after completion of the phosphoethanolamine-containing structure. The completed GPI moiety is then attached to selected proteins via a transamidation reaction in which a C-terminal GPIdirecting signal sequence in the acceptor protein is removed and replaced with the GPI moiety linked, via ethanolamine, to the newly exposed C-terminal amino acid of the protein. Mutants in yeast (guul,gpi8) and mammalian cells (class K) have been identified that appear to synthesize the complete GPI anchor precursor yet lack GPIanchored proteins [7-91. These cells are probably defective in the transamidase. Some years ago we proposed that the GPIbiosynthetic pathway is confined to the cytoplasmic face of the ER membrane with the mature triply mannosylated phosphoethanolamine-containing GPIs being flipped into the exoplasmic leaflet (via a GPI flippase?) for transfer to protein ([ 10,111; see also [12]). This proposal was based on in vitro studies in which freshly synthesized GPIs were found to be accessible to GPI-recognizing membrane topological probes (PI-specific phospholipase C or lectins) in topologically defined membrane systems. Although these data clearly showed that GPI structures could be found in the cytoplasmic leaflet of microsomal membranes, the likely presence of phospholipid and glycolipid translocators in these membranes made it impossible to state with any certainty that GPIs are also synthesized in the cytoplasmic leaflet. It is possible that GPIs are synthesized in the lumenal leaflet of the ER and ‘flipped’ out by lipid translocators, thus becoming accessible to topological probes confined to the cytoplasmic face of the membrane. We recently examined the topological distribution of radiolabelled GPIs in subcellular frac-

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tions prepared from metabolically labelled thymoma cells. These experiments indicated that, although the more mature (mannosylated) GPI structures could be accessed by a membrane topological probe [GPI-specific phospholipase D (PLD)] in crude subcellular fractions (e.g. a 10 000 g pellet obtained from a cell homogenate), these GPIs were inaccessible to the same probe in relatively clean preparations of ER obtained via sucrose-gradient centrifugation (see [lo]). In contrast, similarly ‘clean’ ER vesicles were previously shown to be capable of synthesizing GlcNAc-PI and GlcN-PI from UDP[3H]GlcNAc, and displaying these lipids on the outer (cytoplasmic) leaflet of the vesicle membrane bilayer [lo]. These and other results have led us to propose the model shown in Figure 1. The principle feature of this model that distinguishes it from previous proposals is the compartmentation of the early and late GPI-biosynthetic steps in separate but functionally associated membrane domains (regions A and B; Figure 1). GlcNAc-PI and GlcN-PI are synthesized on the cytoplasmic face of certain regions of the ER (region A) [10,11,13]; we propose that region A contains protein-translocation machinery and the GPI transamidase, but no translocators capable of flipping GPIs. GlcN-PI is transferred (perhaps diffusively or at points of membrane contact) to an ER-associated membrane or ER subdomain (region B) where it is inositol-acylated and elongated by mannosylation and phosphoethanolamine addition. The final feature of the model is that mature GPI structures return to the original transamidase-containing ER (region A) for attachment to protein. We propose that region B contains lipid translocators (flippases) capable of flipping GPI intermediates back and forth across the bilayer [11,14]. Assuming GPI flip-flop in region B, it would be impossible to predict the transbilayer arrangement of the GPI-elongation steps by assaying the availability of GPI-biosynthetic intermediates to membrane topological probes. It could be argued, for example, that GPI mannosylation and phosphoethanolamine-transfer reactions occur on the lumenal face of the ER but that, through the action of region-B lipid translocators, GPI intermediates can be detected (as we and others have shown [ll’lZ]) on the cytoplasmic face. One piece of evidence in support of lumenally oriented elongation reactions is the identification and structural characterization of a candidate GPI mannosyltransferase (the PIG-B


gene product) that appears to be a membrane protein with a large lumenal (putative catalytic) domain [15]. Thus ‘clean’ ER vesicles purified from cell homogenates would essentially consist of regionA membranes. These vesicles would be competent to synthesize GlcN-PI (but, in our experience, unable to elongate it efficiently) and transfer GPI anchors to protein. The vesicles would also be expected to contain mature GPIs (such as those synthesized during metabolic labelling of the cells before preparation of an ER fraction) topologically locked in the lumenal leaflet of the membrane bilayer. Cruder preparations such as the cell homogenates and unfractionated microsomes used by many investigators in studies of GPI biosynthesis in vitm would contain both A and B regions of the ER, functionally linked so as to be able to carry out all the GPIbiosynthetic reactions. The availability of regionB lipid translocators in such crude preparations would also allow GPI-structures (synthesized in vitm from radioactive sugar nucleotides or by metabolic labelling before subcellular fractionation) to be accessed by membrane topological probes. Region B could be the mitochondrion-associated ER subfraction [mitochondrion-associated membrane (MAM)] described by Vance [16]. This subcellular membrane possesses many features characteristic of the ER, but fractionates with mitochondria in differential centrifugation protocols. The MAM is also enriched in a variety of phospholipid-biosynthetic enzymes. The nature of lipid transfers between the MAM, ER and mitochondria is not fully understood but must necessarily occur, for example, in the conversion of phosphatidylserine (PS) to phosphatidylcholine (PC) via PS decarboxylation and PE methylation [ 171.

lntracellular transport GPI-anchored proteins traverse the secretory pathway from their point of assembly, the ER, to their primary cellular location, the cell surface. Low rates of endocytosis provide for long-lived surface expression of these proteins, and the plasma membrane may be realistically considered as the end point of their transport pathway. In contrast, little is known about the subcellular distribution and transport of nonprotein-linked GPIs. GPIs are made in excess of the amount needed for protein modification, and eukaryotic cells contain significant pools of non-

protein-linked GPIs (105-107 molecules per cell depending on GPI structure and cell type) in addition to GPI structures (anchors) covalently linked to protein. Evidence from different experimental systems suggests that a significant fraction of these GPIs may exit the ER and relocate to other organelles, including the plasma membrane. The localization of GPIs to the plasma membrane has implications for models of signaltransduction pathways, particularly those proposed to mediate insulin action [18]. In these models, glycan second messengers are generated via the insulin-stimulated phospholipase Cmediated hydrolysis of non-protein-linked GPIs located in the cytoplasmic leaflet of the plasma membrane. Although a number of ambiguities exist in experimental data purporting to support these ideas, recent work clearly shows that GPIs play an essential role in the stimulation of glycogen synthesis by insulin [ 191. The presence of non-protein-linked GPIs at the plasma membrane is clearly indicated in recent studies [ZO] where we showed that GPIs are constituents of the lipid envelope of influenza virus and vesicular stomatitis virus 0. These viruses acquire their envelope lipids solely from the plasma membrane; the presence of GPIs in the viral envelope therefore indicates that GPIs are plasma-membrane constituents. These experiments, performed in polarized MadinDarby canine kidney epithelial (MDCK) cells, showed in addition that, unlike GPI-anchored proteins that are expressed exclusively at the apical cell surface, the surface distribution of non-protein-linked GPIs is not polarized. The lack of polarity suggests (but does not prove) that non-protein-linked GPIs are primarily located in the cytoplasmic leaflet of the plasma-membrane lipid bilayer. In order to examine the transbilayer distribution of GPIs at the plasma membrane, we prepared topologically defined plasma membrane ‘vesicles’ in the form of VSV particles, and probed for GPI orientation. The VSV envelope is derived from the plasma membrane and has been shown to be generally (although not perfectly) representative of the plasma membrane in terms of phospholipid content and asymmetry [21,22]. Furthermore, importantly, the VSV envelope lacks the ability to promote transbilayer movement of lipids, thus allowing us to probe a static transbilayer lipid arrangement [23,24]. VSV particles were purified from infected C3H]mannose-labelled MDCK cells [ZO], treated with

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trypsin to strip the glycoprotein coat (this treatment did not affect the capsid proteins), and incubated with purified GPI-PLD (obtained from Dr. P. Butikofer, Universitat Bern, Bern, Switzerland). No GPI hydrolysis was observed, except in the presence of detergent. In contrast, GPIPLD hydrolysed approx. 50% of the mature GPIs (and approx. 75% of GPI-biosynthetic intermediates) in the MDCK cell residue recovered after virus infection. Similarly, GPIs synthesized in vitro using thymoma cell lysates or trypanosome membranes were also hydrolysed by GPI-PLD. Thus GPI-PLD is able to hydrolyse membrane-bound GPIs. A potential problem is that the PLD may not have been able to access the viral envelope bilayer, resulting in non-hydrolysis of GPIs. We tested probe access to the bilayer (and also verified intactness of the stripped viral particles) by investigating phospholipid hydrolysis by Bacillus cereus phospholipase C treatment of stripped viral particles: viral PC was completely hydrolysed in the stripped particles, but viral PE (expected to be mainly in the inner leaflet of the viral membrane) was hydrolysed only when detergent was included in the assay. Thus the stripped viral particles are effectively 'naked', and the envelope bilayer is accessible to topological probes. The non-hydrolysis of viral GPIs by PLD can therefore be taken to imply that GPIs are located in the inner leaflet of the viral envelope, i.e. the cytoplasmic leaflet of the plasma membrane. There is very little direct information on the mode of intracellular GPI transport. One strategy to acquire basic information on this problem is to analyse metabolically labelled cells by subcellular fractionation. Using this method we have discovered that even in the shortest metabolic labelling times ( - 5 min) that allow us to generate enough radiolabelled GPIs for detection purposes, the GPIs are already in all the major subcellular fractions that can be derived from a postmitochondrial supernatant, i.e. ER, Golgi and plasma membrane. There appears to be some slight transport selectivity, since membranes that cofractionate with the Golgi marker in our analyses are not as intensely decorated with GPI as 'membranes cofractionating with the ER or plasma membrane. Similar distributions were obtained with cells labelled for 1 h at 37"C, or 1 h at 15"C, a condition known to halt vesicular transport between the ER and Golgi. Most importantly, the distribution of GPIs did not change if the 5 min labelling period was followed

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by a 1 h chase. These results suggest that GPIs are transported by a relatively rapid process that is essentially complete in less than 5 min at 37°C. Moreover, the pulse-chase data suggest that we are dealing with an equilibration process rather than vectorial transport. By these criteria, transport of unattached GPIs appears not to occur by vesicular means, but rather by other mechanisms such as protein-assisted transfer through the cytosol [17]. Our data are similar to those obtained in studies of phospholipid transport where half-times for transit from the ER to the plasma membrane were found to be of the order of 1-2 min [25,26], presumably because of the involvement of non-vesicular transport mechanisms. A potential complication in the interpretation of our subcellular fractionation results is that the precise experimental outcome may have been influenced by GPI redistribution during sample work-up, despite the care taken to maintain all samples at 0°C. However, GPI redistribution during sample work-up would really only be possible if GPI transport in the cell occurred via non-vesicular mechanisms. Thus, although the fractionation experiments may be somewhat misleading as far as the assessment of the rate of GPI transport is concerned, conclusions from these experiments on the transport mechanism remain sound. Membrane proteins, for example, do not redistribute as attested to by our ability to detect organelle-specific marker enzymes in separate subcellular fractions. Nor does cholesterol, a lipophilic molecule that is postulated to use vesicular transport pathways [27]. The only ways in which we would have obtained an ER-restricted GPI distribution in a pulse-labelling experiment are if GPIs were not transported at all (which we know is not the case [20]) or if transport occurred comparatively slowly (tens of minutes) as would be the case for conventional multistep vesicular transport. In related work, using an amine-derivatization procedure to detect cell surface GPIs, Singh et al. [28] reported that GPIs are transported to the cell surface relatively slowly, over a period of several hours, and that transport is reduced under conditions (15°C incubation) where ER-Golgi vesicular transport is blocked. On the basis of these data, Singh et al. suggested that GPI transport from the ER to the cell surface occurs via standard transport vesicles. We propose that Singh et al. may be describing a by-product of transport where GPIs are delivered


rapidly to t h e cytoplasmic face of t h e plasma membrane, then flipped slowly on to the cell surface in a temperature-dependent fashion. A potential flipping mechanism could involve products of multidrug resistance genes, some of which appear to be able to translocate phospholipids a n d glycolipids [29].

Summary The subcellular compartmentation of t h e biosynthetic reactions involved in GPI assembly is likely to be more complex than originally envisaged. T h e model presented in Figure 1 elaborates previous proposals to account for new experimental data. The principal new feature of t h e model is the separation of early and late reactions in GPI biosynthesis in distinct but functionally associated membranes, possibly subdomains of the ER. The observation that biosynthetic lipid intermediates a s well as mature GPIs escape their site of synthesis to populate other cellular m e m b r a n e s has implications for the control of the assembly process and suggests possible cellular functions of GPIs. Current data indicate that the principal m e a n s of intracellular GPI transport is non-vesicular, suggesting a potential role for lipid-transfer proteins in t h e process. The presence of GPIs in t h e cytoplasmic face of t h e plasma m e m b r a n e is particularly significant for models where GPIs have been proposed to act as second messengers in signal-transduction pathways. This work was supported by grants from the National Institutes of Health, the Howard Hughes Medical Institute, and the University of Wisconsin. T h e Figure was prepared by Robin Davies. A.K.M. acknowledges Peter Butikofer for providing GPI-PLD, and A. Dalgleish for stimulation.

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Received 7 March 1997

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