Trafficking and localisation of resident Golgi glycosylation enzymes

Biochimie 83 (2001) 763−773 © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908401013128/REV

Trafficking and localisation of resident Golgi glycosylation enzymes Andrew S. Opat, Catherine van Vliet, Paul A. Gleeson*1 Department of Pathology and Immunology, Monash University Medical School, Melbourne, Commercial Road, Melbourne, Victoria 3181, Australia (Received 19 April 2001; accepted 18 June 2001) Abstract — The localisation of glycosylation enzymes within the Golgi apparatus is fundamental to the regulation of glycoprotein and glycolipid biosynthesis. Regions responsible for specifying Golgi localisation have been identified in numerous Golgi resident enzymes. The transmembrane domain of Golgi glycosyltransferases provides a dominant localisation signal and in many cases there are also major contributions from the lumenal domain. The mechanism by which these targeting domains function in maintaining an asymmetric distribution of Golgi resident glycosylation enzymes has been intensely debated in recent years. It is now clear that the targeting of Golgi resident enzymes is intimately associated with the organisation of Golgi membranes and the control of protein and lipid traffic in both anterograde and retrograde directions. Here we discuss the recent advances into how Golgi targeting signals of glycosylation enzymes function, and propose a model for maintaining the steady-state localisation of Golgi glycosyltransferases. © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Golgi / glycosylation / membrane transport / Golgi localisation / glycosyltransferase / trans-Golgi network

1. Introduction The Golgi apparatus is not only responsible for the exquisitely regulated glycosylation of glycoproteins and glycolipids but is also the hub of the secretory pathway. Glycosylation events mediated by Golgi enzymes are critical for the development of multicellular organisms. While cultured cell lines are largely unaffected by mutations in glycosyltransferases, or drug-induced inhibition of glycosylation, glycosyltransferase deficient mice exhibit lethal or semi-lethal phenotypes [1–3], and a number of diseases are known to be the result of glycosylation deficiencies [4, 5]. Furthermore, carbohydrates are important in cell-cell communication, and for biological activity of many proteins [6]. The large diversity of carbohydrate structures is the product of over 200 different glycosyltransferases residing in the Golgi apparatus. Clones encoding many of these proteins have now been isolated, however, the precise localisation of only a small number of the glycosyltransferase proteins within the Golgi apparatus has been determined. Nonetheless the ultrastructural localisation data of these few glycosyltransferases has provided important clues in trying to figure out how these enzymes specifically reside in the Golgi apparatus. For *Correspondence and reprints. E-mail address: [email protected] (P.A. Gleeson). 1 Address from 1/10/2001: Department of Biochemistry and Molecular Biology, The University of Melbourne.

example, enzymes involved in the synthesis of complex N-glycans show distinct but overlapping distributions that are consistent with their order in the glycoprotein biosynthetic pathway [7]. Of significance is that the enzymes are not segregated into clearly defined subcompartments, but form overlapping concentration gradients across the stack. There must be mechanisms to account for the different distributions of particular glycosylation enzymes across the Golgi stack and, further, such mechanisms must also explain the basis for the overlapping gradients of enzymes across the stack. In addition to mapping the precise localisation of individual glycosyltransferases within the Golgi cisternae, it is also important to appreciate how the large number of glycosyltransferases are organized within the membranes of the Golgi apparatus, and how this relates to the regulation of the synthesis of oligosaccharide structures. Over the past 10 years many studies have attempted to identify signals in Golgi resident proteins responsible for their segregation from secretory traffic and localisation within discrete sections of the Golgi apparatus. Collectively, these studies have been unable to identify specific targeting sequences, but have nonetheless identified regions of the glycosyltransferases that are critical for their compartment-specific localization (see reviews [8–10]. The identification of these Golgi targeting domains was relatively straightforward given the techniques available to cell biologists. However, unravelling the mechanism by which these targeting domains function has proven to be considerably more difficult. Understanding how resident glycosyltransferases are localised within the Golgi,

764 against the flow of soluble and membrane proteins moving through the Golgi en route to other destinations, assumes a knowledge of the mechanism for vectorial movement of cargo across the Golgi stack. New insights concerning the transport of cargo through the secretory pathway over the past 5 years have challenged cell biologists to reassess how retention and segregation of Golgi-resident glycosylation enzymes occurs. It is now clear that the mechanism of targeting Golgi glycosyltransferases is intimately associated with organisation of Golgi membranes and the control of protein and lipid traffic in both forward (anterograde) and reverse (retrograde) directions. Thus, understanding the basis for the localisation of glycosyltransferases to the Golgi apparatus has progressed from the simple notion of ‘defining retention signals of glycosyltransferases’, to one of understanding the biogenesis of Golgi membranes. This review will briefly summarise the data on signals for Golgi localisation and then focus on the issues pertaining to trafficking of proteins through the Golgi and recent data that sheds some light into how the Golgi targeting signals of glycosylation enzymes function. 2. Signals for Golgi localisation All Golgi glycosyltransferases share a common topology and domain structure. They consist of a short amino-terminal cytoplasmic tail, a transmembrane domain, and a lumenal domain which includes a ‘stalk’ followed by the catalytic domain. Despite this common domain structure, regions of homology at the amino acid level are scarce. A study of cloned glycosyltransferases classified 555 sequences into 26 families, according to sequence similarity [11]. However, only seven of these families contained two or more different transferases, and N-acetylglucosaminyltransferase I (GlcNAc TI), II, III, and V (four enzymes involved in N-glycan biosynthesis and utilizing UDPGlcNAc as a donor) were classified into different families indicating no common homology [11]. On the other hand it has become clear that certain glycosyltransferases, e.g., β1,4 galactosyltransferase (β4Gal-T), are part of a multigene family with short regions of homology likely to represent a common substrate binding domain [12]. A number of conserved motifs have also been detected in sialyltransferases, which appear to be important in binding to the nucleotide sugar donor substrate [13]. Due to this very limited homology between glycosyltransferase, no peptide motifs which might facilitate localisation have been identified by sequence comparison. Instead, the general strategy for investigating targeting signals within the glycosyltransferases has been to construct deletion mutants, or fusion proteins with non-Golgi proteins. Initially the most intensely studied glycosyltransferases were the trans-Golgi/trans-Golgi network (TGN) enzyme, α2,6 sialyltransferase (ST6Gal I), the trans-Golgi

Opat et al. enzyme β4Gal-T1, and the medial-Golgi enzyme, GlcNAc-TI (see reviews [8, 9]). Numerous other glycosyltransferases have now also been examined, including a number of yeast and plant Golgi glycosyltransferases [8, 14, 15]. Together, these include enzymes localised to the cis-, medial- and trans-Golgi/TGN. The overriding conclusion from the analysis of glycosyltransferase chimeras is that the transmembrane domain of many different glycosyltransferases provides a dominant localisation signal (see reviews [8, 9]). In addition, a number of studies have shown contributions from the lumenal domain and cytoplasmic tail, suggesting that there may be multiple signals involved in the specific localisation of these Golgi enzymes [8, 9]. It is likely that the mechanisms that retain proteins in the Golgi apparatus are highly conserved through evolution, as mammalian glycosyltransferases have been expressed in yeast and plant cells, and are correctly localized in each case to the Golgi [16–18]. Despite this conservation, a number of subtle differences have been observed between different studies. Notably some native glycosyltransferases and chimeric proteins have exhibited slightly different localisations in different cell types [19–21]. Another potential difficulty in comparing the fine details of localization between studies is that different proteins have been used as fusion partners in the construction of chimeras. A critique of the cell type and fusion partner differences in these studies is given in the excellent review by Colley [8]. Although these localisation studies have been informative, in most cases it is not clear whether the Golgilocalised glycosyltransferase chimeras are targeted correctly so they can function appropriately in vivo. This is an important question as there may be different regions of the glycosyltransferase responsible for Golgi localisation and for the correct positioning of the membrane protein within Golgi membranes in relation to the other enzymes in the glycosylation pathway. Two recent studies have addressed this issue and have analysed the oligosaccharide products produced by cells expressing the chimeric glycosyltransferase molecules. A study by Grabenhorst and Conradt [22] mapped the functional positions of nine different glycosyltransferase chimeras; these investigators showed that the cytoplasmic, transmembrane domain and stem regions of a number of different enzymes contain the necessary information for targeting not only to the Golgi but also to the correct functional subcompartments. However, the relative contribution of stem and transmembrane domains to the correct functional localisation was not assessed in this study. In a recent study from our laboratory we showed a GlcNAc-TI chimera, where the transmembrane domain and cytoplasmic region of GlcNAc-TI was replaced with corresponding regions derived from the transferrin receptor, is able to rescue the GlcNAc-TI glycosylation defect of Lec1 cells, demonstrating that the chimeric enzyme containing only the lumenal sequences

Trafficking of Golgi glycosyltransferases of GlcNAc-TI is functionally active in vivo [23]. Detailed analysis of the functional activity of other glycosyltransferase chimeras would also be worthwhile. One of the ongoing difficulties in defining bona fide Golgi targeting signals of glycosyltransferases is the potential of the glycosyltransferase chimeric molecules to form complexes with other endogenous Golgi membrane components. This raises the possibility that Golgi targeting information may be present in other components of the complex. Thus, the Golgi localisation of some of the glycosyltransferase chimeras may be due solely to sequences that promote their interaction with existing membrane complexes. This could be especially pertinent to the ability of the lumenal domain of some of the glycosyltransferases to target chimeras to the Golgi. For example, GlcNAc-TI has been shown to exist as high-molecular mass complexes and analysis of membrane-bound GlcNAc-TI chimeras indicates that the formation of high molecular mass complexes does not require the transmembrane domain and cytoplasmic sequences of GlcNAc-TI [23]. In fact, a soluble form of GlcNAc-TI, containing the stem region and catalytic domain, accumulated in the Golgi prior to secretion [23]. It is likely that the inclusion into the high molecular mass complexes accounts for the Golgi localisation of both the membrane-bound GlcNAc-TI chimera and soluble GlcNAc-TI. 3. Mechanism of localisation of glycosyltransferases Based on the observation that the transmembrane domain of glycosyltransferases contains a dominant localisation signal, two models were initially proposed and have since been widely discussed to explain how resident enzymes may be localised to the Golgi apparatus. One model is based on lipid bilayer sorting and the other on oligomerisation of Golgi resident proteins. Both models initially favoured mechanisms in which Golgi glycosyltransferases were prevented from entry into forward moving transport vesicles and thereby are actively retained in one Golgi region. This idea was based in part on the finding that the late-acting enzymes sialyltransferase and galactosyltransferase were found to be localised to the trans-Golgi and TGN without any evidence of recycling over the plasma membrane [20, 24]. Although it is now clear that active retention is not the sole basis for localisation, both models are still nonetheless relevant to a more contemporary view of localisation mechanisms. 3.1. Lipid bilayer sorting The lipid bilayer sorting model, proposed by Bretscher and Munro [25] is centred around three observations; firstly, mutational analysis of the transmembrane domains of Golgi enzymes failed to identify individual residues or sequence motifs involved in localisation indicating that a

765 more general property of the hydrophobic region was relevant; secondly, lipid bilayer composition is not constant throughout the secretory pathway; and thirdly, that Golgi localised proteins tend to have a shorter transmembrane domain than plasma membrane proteins [26, 27]. In mammalian cells the cholesterol content increases from ER membranes to the plasma membrane [28] and the increase in cholesterol content results in a thickening of the membrane bilayer [29]. Munro suggested that the thinner bilayer of the Golgi, compared with the sphingolipid and cholesterol-rich post-Golgi membranes, may preferentially retain resident enzymes which generally have shorter transmembrane domains. Indeed, replacement of the 17 amino acid ST6Gal I transmembrane domain with 17 leucines does not alter its Golgi localisation [30]. On the other hand, if the transmembrane domain is increased to 23 leucines ST6Gal I is found on the plasma membrane [30]. Shortening the transmembrane domain of a plasma membrane protein from 23 to 17 residues also reduces its trafficking to the cell surface, resulting in accumulation in the Golgi apparatus [31]. While the transmembrane domain of Golgi proteins do tend to be shorter than those of plasma membrane proteins, there are exceptions, suggesting that either Golgi localisation signals may be dependent on particular characteristics of the hydrophobic amino acids of the transmembrane domain rather than just in its length, or that we do not understand how the sequence is folded. Although the lipid sorting model is very appealing, various studies have shown that the transmembrane domain does not fully account for the localisation of Golgi enzymes. Colley and colleagues identified two isoforms of ST6Gal I which differ by a single amino acid at residue 123 in the lumenal domain [32]. The Cys isoform of ST6Gal I is retained in the Golgi more efficiently than the Tyr isoform [32]. Further, the Cys isoform of ST6Gal I does not appear to be as sensitive to changes in the length of the transmembrane domain indicating that the localisation involves lumenal sequences as well as the transmembrane domain [33]. Additionally, in a study on the localisation of GlcNAc-TI chimeras expressed at modest levels in stable cell lines, it was clearly demonstrated that efficient localisation of GlcNAc-TI required a significant contribution from the lumenal domain and a minor additional contribution from the cytoplasmic tail [34]. 3.2. Oligomerisation or kin recognition The aggregation model was first proposed by Machamer [35], who suggested that glycosyltransferases are induced to form aggregates as they reach the correct Golgi compartment. At the time, Machamer proposed that the interaction between the transmembrane domains of glycosyltransferase and lipid bilayers of Golgi membranes may be the driving force for oligomerisation. An extension of the aggregation model was put forward by Warren and

766 colleagues who suggested that different enzymes of a particular Golgi compartment interact with each other (kin recognition) forming large hetero-oligomeric structures [36]. Nilsson et al. [37] showed that two enzymes of the medial-Golgi interact with each other as the addition of an ER retention motif to the GlcNAc-TI cytoplasmic tail not only caused GlcNAc-TI to localise to the ER but also partially retained another medial-Golgi enzyme, namely α-mannosidase II (MannII), within the ER. In contrast the trans-Golgi enzyme, β1,4GalT, was not relocated to the ER in these experiments, indicating specific association of enzymes from the same compartment. Although this ‘kin-recognition’ interaction was initially suggested to occur via the transmembrane domain it was subsequently shown that the stem region of GlcNAc-TI, and not the transmembrane domain, was responsible for association with MannII [38]. Recent studies in our laboratory have detected high molecular mass complexes of the two medial-Golgi enzymes, GlcNAc-TI and GlcNAc-TII, which is consistent with the suggestions of Warren and colleagues, and furthermore, directly demonstrates that medial-Golgi enzymes exist as complexes within Golgi membranes [23]. As for kin recognition, the formation of high molecular weight complexes of GlcNAc-TI was shown to be mediated via the lumenal sequences [23]. A similar finding has recently been reported for GlcNAc-TV [39]. Nonetheless, whereas kin recognition required the presence of charged residues in the stem region of GlcNAc-TI [38], studies from our laboratory have shown that these residues were not important for the ability of GlcNAc-TI chimeric molecules to be either localised within the Golgi, included into high molecular mass complexes, or functionally active in vivo. The most likely explanation for this difference is that Nilsson et al. [38] assessed the importance of the charged residues in chimeric molecules containing the stem of GlcNAc-TI in the absence of the GlcNAc-TI lumenal catalytic domain, i.e., the mutated chimeric molecules contained only the stem region of GlcNAc-TI. If the formation of high molecular mass complexes involving medial-Golgi enzymes involves multiple contacts between the protein molecules of the complex, mutating the charged residues of the GlcNAc-TI stem may not interfere with contacts with other regions of the lumenal domain. Furthermore β1,4Gal-T and α1,2fucosyltransferase were detected as monomers and dimers under the same extractions conditions that showed GlcNAc-TI as high molecular mass complexes, indicating that late Golgi enzymes behave differently to medialGolgi enzymes [23]. Using the kin recognition assay, Munro [31] could not detect a specific interaction between the two trans-Golgi enzymes ST6Gal I and GalT (within the ER environment), again consistent with differences in the behaviour of late Golgi and medial-Golgi enzymes. Colley and co-workers [40] have made the important observation that ST6Gal I form insoluble oligomers at a

Opat et al. pH of 6.3, corresponding to the pH of the late Golgi, and furthermore the ability to form oligomers correlated with stable Golgi localisation. The pH dependence of ST6Gal I complex formation suggests that the glycosyltransferases of the late Golgi may only form complexes on arrival at the late Golgi, in contrast to the medial-Golgi enzymes which can interact at the higher pH of the ER or early Golgi environment [23, 37]. Collectively, these studies indicate that glycosyltransferases from different Golgi compartments form high molecular mass complexes and that the inclusion into such complexes increases the efficiency and stability of Golgi localisation. There is increasing evidence for the existence of multi-enzyme complexes within the Golgi. The yeast enzyme Mnn9p co-precipitates with a number of proteins which bear homology to glycosyltransferases and activity assays on the Mnn9p immunoprecipitates indicated the presence of multiple mannosyltransferase activities [41]. Recent elegant studies on sphingolipid synthesis, employing both in vitro biochemical approaches and in vivo FRET analysis, has also revealed that two glycosyltransferases involved in sequential steps of glycolipid synthesis are physically associated [42]. In summary, it is clear that there is no discrete signal involved in Golgi localisation. Instead, it is likely to involve disparate regions of the molecule. Two or more independent signals, each involving distinct interactions, may act together to mediate efficient Golgi localisation. The presence of multi-enzyme complexes is likely to be functionally relevant in the regulation of glycosylation, and may also play an indirect role in the precise localisation of the complexes within Golgi membranes. 4. Golgi recycling and mobility of Golgi glycosyltransferases Immunolocalisation provides a snap-shot of the distribution of a glycosyltransferases at any moment in time and thus represents a steady-state distribution. It gives no information on whether the proteins are actively retained or constantly moving. Analysis of the mobility of Golgi enzymes within the membranes of living cells using green fluorescent protein tagged resident Golgi glycosyltransferases, showed that the enzymes could diffuse rapidly and freely in Golgi membranes [43]. This study revealed that enzymes from both the trans- and medial-cisternae are highly mobile, indicating that Golgi targeting and retention does not depend on protein immobilisation. It would also suggest that any differences in the oligomerisation of medial- and trans-enzymes has little effect on their mobility within the lipid bilayer. However, one caveat of the study by Cole et al. [43] is that it used high levels of the GFP fusion proteins in transient expression systems. High levels of the Golgi enzymes may saturate the oligomerisation mechanisms and it is possible that excess fusion

Trafficking of Golgi glycosyltransferases protein may exist in a different physical state to the endogenous protein. Significantly, the mobility of GFP tagged GlcNAc-TI when expressed at more modest levels in stable cell lines appears to be considerably lower than the mobility of GFP fusion of Cole et al. [43], as indicated by diffusion rates following photobeaching (G. Banting, personal communication). Additional studies to compare the relative mobility of resident Golgi enzymes over a range of expression levels would be well worthwhile. Nonetheless, it is clear that Golgi enzymes are not fixed permanently in one membrane compartment. Cis-Golgi residents of yeast cells have been shown to acquire modifications of the late Golgi [44] and there is compelling evidence that resident Golgi glycosyltransferases have the ability to recycle from the Golgi to the ER [45]. Consideration of the trafficking and recycling pathways of Golgi enzymes have now became a major focus of investigation. 5. Models for transport through the secretory pathway Integral to understanding the basis of the steady state distribution of glycosyltransferases is the mechanism for movement of protein cargo through the Golgi stack. Anterograde (forward moving) vesicular transport and cisternal maturation are two alternative models of cistrans Golgi transport that have been hotly debated over the past few years. The anterograde vesicular transport model predicts that resident proteins are specifically retained, while cargo molecules will move forward in transport vesicles which bud and fuse among static cisternae. The cisternal maturation/retrograde transport model predicts that cargo molecules move through the stack passively as the cisternae move forward, with no transport vesicle involved in this forward movement, while resident proteins are recycled by retrograde transport to establish differential concentrations across the stack. Mechanisms involving active retention of glycosyltransferases can only apply in the situation where forward movement of cargo occurs via vesicular transport; if cargo is moved through the Golgi stack solely by cisternal maturation then retention mechanisms are not relevant. Clearly, understanding Golgi transport is fundamental to determine the relative contribution of retention and recycling in the steady-state localisation of Golgi glycosyltransferases. As this is a central issue to understanding the localisation of Golgi enzymes it is discussed in more detail below. 5.1. Vesicular transport The vesicular transport model for cargo movement defined the Golgi cisternae as stable structures, with proteins being moved between cisternae in small transport vesicles [46] (figure 1A). The idea of stable cisternae

767 structures appeared very attractive as it was compatible with the finding that different Golgi cisternae housed different sets of enzymes and, in addition, vesicular transport was consistent with the appearance of vesicles associated with Golgi membranes [47]. The vesicles were found to bud from all cisternae of the stack and were associated with a specific protein coat, known as COPI (COat Protein I or coatomer). The COPI coat is responsible for driving the budding of these Golgi vesicles [48]. This coat consists of seven subunits which are recruited en bloc from the cytoplasm to Golgi membranes by the GTP bound form of the small GTPase, ARF1 [49]. The nucleotide exchange factor responsible for activating ARF1 is sensitive to the fungal metabolite brefeldin A, and brefeldin A treatment of cells results in the inactivation of ARF1, the subsequent cessation of COPI budding and the inhibition of anterograde transport [50]. Based on these and other observations from in vitro transport assays, COPI vesicles were proposed to be mediators of anterograde traffic between Golgi cisternae. Over the past 4–5 years a number of serious difficulties have emerged with the forward vesicular transport model. It was originally assumed that COPI coated vesicles derived from the Golgi membranes represented forward moving transport vesicles. However, there is now considerable evidence that COPI vesicles mediate retrograde transport from the Golgi to the ER [51]. Another essential component of vesicular transport is the SNARE proteins, integral membrane proteins that mark both the transport vesicle and the acceptor membrane compartment, and which contribute to the specificity of vesicle docking and fusion. Yeast SNAREs appear to define different compartments of the secretory and endocytic pathway, but there are insufficient members of the ‘classical’ SNARE family in the yeast genome to account for anterograde transport steps through the Golgi of yeast cells [52]. 5.2. Cisternal maturation Cisternal maturation is a modified version of cisternal progression, first proposed in the 1950s and later refined by Morré [53], which suggested that the Golgi is a type of escalator with cisternae forming from ER-derived vesicles at the cis face of the Golgi and moving sequentially as discrete units through the stack towards the trans face where the membranes are then utilised in transport vesicles (figure 1B). This model was supported by observations of scale assembly and transport in algae [54]. However, during the 1960s and 1970s it was realized that the Golgi stack consisted of distinct compartments each with a different composition of carbohydrate modifying enzymes and other resident proteins [55]. This conceptual advance was difficult to reconcile with the cisternal maturation model and subsequently many (especially animal) cell biologists discarded the model on the basis that algal scale transport was somehow an exception [55].

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Figure 1. Models for transport of proteins through the Golgi apparatus. A. The vesicular transport model is based on cargo moving in vesicles through a series of stable cisternae while resident Golgi proteins are retained by exclusion from these transport vesicles. CGN, cis-Golgi network. B. The cisternal maturation model. In this model, cisternae are formed de novo at the cis face and move sequentially as discrete units through the stack towards the trans face where the membranes are then utilised in transport vesicles. Retrograde vesicular transport from the late Golgi compartments maintains the differences in the distribution of resident proteins throughout the stack.

The discovery of retrograde or backward transport was a pivotal finding. Originally discovered as the mechanism for retrieval of resident ER proteins that had ‘leaked out’ of the ER and moved down the secretory pathway [56], it is now clear that proteins from much later compartments can also be retrieved. For example, a chimeric protein consisting of the lumenal domain of the temperaturesensitive vesicular stomatitis virus (VSV) G protein folding mutant VSVGts045, and the transmembrane domain and tail of the TGN38 protein, is recycled to the ER after reaching the TGN [57]. Furthermore, some bacterial and plant toxins, such as shiga toxin and ricin, are internalised at the plasma membrane, then traverse the secretory pathway backwards to the ER, before being translocated into the cytoplasm where they exert their effects [58, 59]. This retrograde transport pathway has been followed by observing the sulphation (TGN modification) and N-glycosylation (ER modification) of internalized toxins before they appear in the cytoplasm [58, 60]. Thus, it appears that there are endogenous retrograde transport mechanisms along the entire secretory pathway.

The existence of a retrograde vesicular transport pathway threw new light on the cisternal progression model. Retrograde transport provided a key to the generation of differences in the distribution of Golgi resident proteins which would overcome the major conceptual difficulty with this model [52, 61]. Thus, the selective delivery of resident Golgi proteins by retrograde transport could result in the enrichment of particular glycosyltransferases in specific compartments and thereby generate cisternae with different compositions of Golgi resident proteins (figure 1B). Forward transport of intact cisternae could occur by the de novo synthesis of new cisternae at the cis face, paired with consumption of membrane at the trans most cisternae by production of vesicles. Evidence for de novo formation of cisternae has come from following the movement of molecules along the secretory pathway of living cells by time-lapse imaging using GFP chimeras [62]. Strikingly, such studies have observed GFP tagged transport intermediates, derived from the ER, move towards the Golgi and spontaneously generate membranes resembling the cis-Golgi network. The continuous forma

Trafficking of Golgi glycosyltransferases tion of new cisternae would represent the first step in the process of cisternal maturation. Similar to the scales in algal cells, procollagen in mammalian cells represents a supermolecular structure (1.5 nm diameter, 300 nm long rods) which would be too large to fit into Golgi transport vesicles (60–90 nm). Bonfanti et al. [63] followed the transport of a synchronized wave of newly synthesed procollagen by immunoelectronmicroscopy. The procollagen aggregates appeared to exit the ER in elongated tubules, which fused with each other at the cis face of the Golgi to form a new cis-Golgi cisterna. Procollagen was observed traversing the Golgi stack in the cis to trans direction without leaving the Golgi cisternae. Serial sections of numerous Golgi stacks failed to detect tubules containing procollagen, indicating that procollagen aggregates are transported through the Golgi by cisternal maturation [63]. A fundamental difference between the two models of transport across the Golgi stack is the differential movement of resident and cargo proteins. However, extreme views of either model are hard to reconcile with all the available data. For example, cisternal progression fails to adequately explain the origin of the TGN, an apparently steady-state compartment containing resident proteins which behaves quite differently from the Golgi stack cisternae after treatment with brefeldin A. As highlighted by Pelham and Rothman [64], the rate of transport of collagen through the Golgi stack is very slow compared to the transport rate of normal cargo; if cisternal progression was the only transport mechanism then the kinetics of transport should be identical in all cases. If cisternal progression is the predominant form of anterograde transport then the resident proteins would have to be sorted and concentrated in retrograde transport vesicles. Lanoix et al. [65] have analysed an uncoated vesicle preparation and detected up to a 10-fold concentration of MannII compared with donor membranes. On the other hand, by immunoelectron microscopy Orci et al. [66] did not detect a concentration of either MannII or GlcNAc-TI in buds or vesicles compared with Golgi cisternae. The discrepancy between the biochemical and immunolocalisation studies in the levels of Golgi resident enzymes is an important issue that needs to be resolved. 5.3. Dual transport systems across the Golgi stack Vesicular transport and cisternal progression are not mutually exclusive and may in fact be occurring simultaneously [64, 67, 68]. Rothman and colleagues have proposed that ‘percolating’ COPI vesicles may provide a ‘fast track’ for cargo transport through the Golgi [68], whereas cisternal flow may be considerably slower in moving cargo across the stack. Indeed, electron microscopic data suggests that COPI vesicles, budding from many levels of the Golgi stack, carry both anterograde and retrograde-directed cargo [67]. As the entry and exit sites

769 of the Golgi are locked, percolating vesicles up and down the stack would result in net movement of cargo in the cis to trans direction. COPI derived vesicles appear to highly constrained near their budding sites by molecular tethers which may limit the transfer of cargo to adjacent cisternae in the stack and thereby overcome the requirement for multiple sets of specific SNARE complexes [69]. Such a model would also provide the ability to segregate anterograde cargo and resident Golgi enzymes between fast and slow moving transport carriers. Protein aggregates too large to enter COPI vesicles, and unable to form the recently described megavesicles [70], would transit via the slow kinetics of cisternal progression. Therefore, there could be multiple levels at which Golgi enzymes are segregated from cargo and from each other. 6. Trafficking of Golgi glycosyltransferases How do glycosyltransferases traffic through the Golgi and what is the precise role of either lipid-mediated sorting or oligomerisation in this process? We still do not have a clear answer to this question. It is possible that the cis-Golgi enzymes are concentrated largely by a recycling mechanism early in the Golgi. Late Golgi enzymes may be restrained from forward transport. On the other hand, understanding the basis for steady-state concentration within the medial-Golgi is more difficult to reconcile. The trafficking of medial-Golgi enzymes is important in understanding precisely how the asymmetric gradients of glycosyltransferases are maintained throughout the Golgi stack. Our recent data on the sialylation rates of a modified GlcNAc-TI bearing an N-glycan site on the C-terminus, shows that GlcNAc-TI is not actively retained for long periods within the Golgi stack, but rather moves down the stack to the TGN and is fully sialylated within 1 h of synthesis [71]. The rate of sialylation of the GlcNAc-TI is comparable with transport rates of cargo molecules, however, it does not distinguish between the vesicular or cisternal progression modes of anterograde transport. The sialylation of N-glycosylated GlcNAc-TI, together with the steady-state localisation within the Golgi stack, indicates that this enzyme recycles from the TGN. Retrograde pathways have been described from the TGN in vivo and indeed Golgi resident proteins have been shown to cycle through the ER [45]. However, the rate of retrograde transport of Golgi residents to the ER is comparatively slow. Given the rapid rate of sialylation of modified N-glycosylated GlcNAc-TI it seems unlikely that direct retrograde transport from the TGN to the ER could account for the medial-Golgi localisation, of this protein. Rather our data on the rate of sialylation indicates that the bulk of the GlcNAc-TI is likely to be recycled from the TGN by intra-Golgi retrograde transport [71]. How might the steady-state distribution of GlcNAc-TI to the medial-Golgi be established? The rate of sialylation

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Figure 2. Model for the localisation of medial and late Golgi resident enzymes. Medial-Golgi enzymes (shown in purple) form large oligomers prior to arrival at the Golgi, and are excluded from ‘percolating’ COPI transport vesicles and move though the Golgi stack by cisternal progression. At the TGN, medial-Golgi enzymes are recruited into retrograde transport carriers and returned to the medial-Golgi. In constrast, late Golgi enzymes (shown in green) may move through the Golgi stack by rapid anterograde transport and arrive quickly at the TGN. Once in the low pH of the TGN, late Golgi enzymes may form complexes which further restrict their forward movement. We suggest, that due to lack of signals or the selective partitioning into distinct lipid domains, late Golgi enzymes do not efficiently recycle and are thereby concentrated in the TGN. The complexes shown may either be homo- or heteroprotein aggregates. CGN, cis-Golgi network.

of the modified N-glycosylated GlcNAc-TI strongly suggests that specific features of retrograde transport from the TGN are critical in establishing the gradient of GlcNAc-TI activity throughout the Golgi stack [71]. It is known that COPI vesicles bud from all levels of the pathway and that COPI is required for intra-Golgi transport in vitro [67]. However, our understanding of intraGolgi retrograde recycling remains rudimentary. Transport intermediates containing resident Golgi enzymes, including GlcNAc-TI, have been shown to fuse preferentially with the cis-Golgi compartment [72]. But retrieval to the cis-Golgi cisternae would not explain how GlcNAc-TI

concentrates within the medial-Golgi. One possibility is that there is selective retrograde transport of GlcNAc-TI from the TGN to the medial-Golgi, but it is unlikely that there is sufficient machinery available, e.g., sets of cognate SNARE molecules, to account for specific intraGolgi transport pathways at every level of the stack [52, 64]. Another possibility proposed by Glick et al. [61] is that there is competition between medial-Golgi enzymes such as GlcNAc-TI and the cis and trans-residents for packaging into vesicles, which could then account for their asymmetric distribution across the stack. This competition could be mediated via a lipid sorting mechanism

Trafficking of Golgi glycosyltransferases [10] to allow selective recruitment of Golgi enzymes into vesicles based on the thickness of the vesicle membrane. Alternatively, the transmembrane sequence of Golgi enzymes may be recognised by a specific retrograde receptor, analogous to the transmembrane-dependent recycling of sec12 by Rer1p from the cis-Golgi to the ER [73]. A further possibility is that medial-Golgi enzymes are efficiently recycled from the TGN in a common pathway back as far as the medial-Golgi cisternae, but then are largely excluded from further retrograde transport, again based on differences in lipid composition between different membrane domains. The finding that medial-Golgi enzymes can be slowly returned to the ER would simply reflect some leakiness in this retrograde transport system. Based on the above discussion a model is shown in figure 2 depicting the trafficking and differential localisation of medial and late Golgi enzymes. We suggest that medial-Golgi enzymes form large complexes early in the biosynthetic pathway, are excluded from anterograde transport vesicles, and move through the Golgi stack by cisternal progression. In contrast, as ST6Gal I requires the low pH of the late Golgi to form high molecular oligomers, this enzyme may be able to move rapidly through the Golgi stack by anterograde vesicular transport and arrive quickly in the TGN. Once in the late Golgi, the formation of large ST6Gal I complexes would stabilize these enzymes and, together with the lack of positive transport signals, restrict movement in the forward direction. In contrast, on arrival at the late Golgi, medial-Golgi enzymes are recruited into retrograde transport vesicles, via specific receptors or lipid mediated sorting, and selectively returned to the middle cisternae. If the latter mechanism was to involve the retrograde transport of lipid domains, rather than the specific cargo recruitment into transport vesicles, then large aggregates could readily be included in tubulo-vesicular transport carriers. 7. Outstanding issues that need to be resolved It is clear that the anterograde and retrograde trafficking pathways of Golgi residents underpin the steady state distribution of glycosyltransferases across the Golgi stack. A more detailed analysis of these trafficking pathways is now required. For example, very little is known about intra-Golgi retrograde transport and the specificity of these intra-Golgi transport steps need to be defined. Is the bulk of Golgi resident enzymes recycled in COPI vesicles or are other populations of retrograde transport vesicles important in this process, such the non-COPI vesicles described by Girod et al. [74]. The signals for inclusion (and exclusion) into retrograde vesicles also need to be identified (i.e., how do resident Golgi enzymes know when and where to recycle?) We still do not have a clear understanding of how the transmembrane domain of glycosyltransferases acts as a localisation signal. Further

771 studies are required to determine if the transmembrane domains of resident proteins have the ability to partition into discrete lipid domains within the membranes of the Golgi. The consequence of aggregate formation in the trafficking of glycosyltransferases needs to be explored more fully, particularly the identification of components within the complex that may be important in mediating recycling. Increasing knowledge of the structures of Golgi glycosyltransferases [75] should also give some insight into the potential of physical association between enzymes acting sequentially within the pathway. Together these different approaches should provide a deeper understanding of the organisation and localisation of the glycosylation enzymes within Golgi lipid bilayers.

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