Subcompartmentalizing the Golgi apparatus

Subcompartmentalizing the Golgi apparatus Manojkumar A Puthenveedu1 and Adam D Linstedt2 The subcompartmentalized structure of the Golgi apparatus contributes to efficient glycosylation in the secretory pathway. Subcompartmentalization driven by maturation relies primarily on constant and accurate vesicle-mediated local recycling of Golgi residents. The precision of this vesicle transport is dependent on the interplay between the key factors that mediate vesicle budding and fusion — the coat proteins and the SNARE fusion machinery. These alone, however, may not be sufficient to ensure establishment of compartments de novo, and additional regulatory mechanisms operate to modify their activity. Addresses 1 Department of Psychiatry, UCSF, San Francisco, California 2 Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA Corresponding author: Linstedt, Adam D ([email protected])

Current Opinion in Cell Biology 2005, 17:369–375 This review comes from a themed issue on Nucleus and gene expression Edited by Vivek Malhotra and Mike Yaffe Available online 21st June 2005 0955-0674/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2005.06.006

Introduction The stability and functional diversity of many newly synthesized proteins in the secretory pathway depend on accurate glycosylation reactions carried out by enzymes located in the Golgi apparatus. The mammalian Golgi apparatus has evolved into a subcompartmentalized structure that optimizes these reaction conditions. This review discusses current views regarding the significance of this subcompartmentalization and our existing knowledge of the mechanisms that underpin it.

Subcompartmentalization ensures optimal processing conditions in the Golgi apparatus Addition of glycan side chains is an ordered process in which each step depends on successful completion of the previous steps. The localization of the enzymes responsible reflects this. Golgi enzymes are present in biochemically distinct membranous compartments termed cisternae that are stacked in a directional manner. Early-acting enzymes are enriched in cis cisternae, and later-acting enzymes are successively enriched in medial and trans cisternae. This resembles an assembly line, www.sciencedirect.com

allowing transiting proteins to encounter the enzymes in a sequential manner. The relative localization of Golgi enzymes with respect to one another influences their access to substrates and dictates the structure of glycans formed in vivo [1]. The extent to which sequential exposure increases the efficiency of processing, however, is not clear. In vivo, Golgi subcompartmentalization can be destroyed by forced redistribution of Golgi enzymes to the endoplasmic reticulum (ER). Under these conditions glycosylation is severely impaired even though the enzymes themselves remain active [2]. It is noteworthy, however, that redistributed Golgi enzymes become significantly diluted in the comparatively vast ER membrane. Furthermore, when substrates are exposed to a mixture of enzymes in vitro, glycosylation eventually proceeds to completion, albeit slowly [3]. Thus, in addition to ensuring sequential exposure, the distribution of Golgi enzymes into subcompartments might allow each subcompartment to achieve the high concentrations of individual sets of enzymes necessary for efficient activity. Subcompartmentalization also allows pH, lipid and ionic gradients across the Golgi stack. With respect to processing, such gradients may be advantageous because, for example, cis-Golgi enzymes can operate in optimized conditions that are distinct from those that are optimal for trans-Golgi enzymes. In sum, subcompartmentalization probably ensures optimal conditions for biosynthetic processing in vivo by establishing sequential substrate exposure, optimized enzyme concentrations and distinct reaction environments. As proteins transit the Golgi apparatus rapidly, leaving only a limited time for contact with enzymes, the high efficiency of processing conferred by subcompartmentalization is important to prevent major changes in glycosylation profiles of biosynthetic proteins.

Subcompartmentalization depends on accurate inter-cisternal vesicle transport mediated by coats and SNAREs Two models have been proposed to explain how Golgi subcompartmentalization is maintained. The first, termed the stable compartments model, proposes that the cisternae are stable entities containing enzymes, and that vesicles selectively transport transiting proteins forward through the Golgi [1]. The second model, termed the maturation model, proposes that Golgi cisternae constantly mature into later compartments by disposing of their enzymes while accepting a new cohort of enzymes from distal cisternae [4]; the transport of enzymes between cisternae occurs via active sorting into vesicles. A cargo molecule may thus be exposed sequentially to Current Opinion in Cell Biology 2005, 17:369–375

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enzymes while traveling forward without ever leaving the cisterna it originally entered. Substantial support exists for both these models, which, although conceptually distinct, are not mutually exclusive [5]. Irrespective of this, however, the key event that maintains subcompartmentalization is the accurate transfer of specific components between cisternae. This begins with coat-protein-mediated sorting of the required proteins into the vesicles that form at the donor compartment. Simply put, proteins that have affinity for the coat proteins will be enriched in a budding vesicle, whereas others will be relatively de-enriched. There are two distinct coats in the early secretory pathway, both of which significantly impact Golgi subcompartmentalization [6]. The coatomer protein II (COPII) coat, formed by the sequential recruitment of GTPbound Sar1, the Sec23/Sec24 complex and the Sec13/ Sec31 complex, mediates ER export by forming vesicles from the ER membrane. The coatomer protein I (COPI) coat mediates most of the vesicle transport between Golgi cisternae. The GTPase Arf1 initiates COPI vesicle formation, as it recruits the seven COPI subunits required to form vesicle buds. In both cases, budding is followed by GTP hydrolysis by the respective GTPases, resulting in uncoating of the vesicles. The vesicles are then targeted for specific fusion with their acceptor compartments, which occurs via the formation of a complex between a vesicle-localized v-SNARE and a target-localized t-SNARE [1]. The tight four-helical SNARE bundle that forms pulls the vesicle and target membranes towards each other and provides the energy for rearrangement of lipid bilayers during fusion. It also contributes to the requisite specificity for fusion, as it relies on specific recognition and binding of cognate v- and t-SNAREs [7]. Interactions between coats and SNAREs ensure the fusogenic potential of the vesicle at the time of budding itself. Although the COPII and the COPI coat complexes are structurally and functionally distinct, both act to concentrate v-SNAREs in the vesicle. Mutational analysis of the COPII coat has identified distinct binding sites for various proteins, including SNAREs, in its sec24 subunit [8]. Further, structural studies of SNARE– sec24 interactions suggest that the sec24-binding motifs on the SNAREs might be buried in the four-helical SNARE bundle that is formed during fusion. This results in sec24 having a higher affinity for free (and hence fusogenic) SNAREs [9,10]. In the case of the COPI coat, SNAREs might form part of the machinery that recruits coat proteins and initiates vesicle budding. Golgilocalized SNAREs bind the GTPase activating protein ARFGAP and then engage the GTPase Arf1 [11,12]. Binding of SNAREs to activated Arf1–GTP might help to stabilize the latter on the membranes to recruit COPI coat components. Inclusion of v-SNAREs into the vesicle may Current Opinion in Cell Biology 2005, 17:369–375

then be mediated by their direct interaction with coat proteins [11]. Thus, sorting of fusion factors by the coat determines the fate and target of the vesicles, and impacts protein transfer between compartments.

A subcompartmentalized Golgi may be generated de novo from the ER Although the exact extent to which maturation contributes to forward transport of cargo in vivo is still unresolved, recent evidence suggests that constant maturation is critical for maintaining Golgi subcompartmentalization. Perhaps the most relevant to the purpose of this review are the experiments that analyze the relationship of the Golgi to the ER. Blocking ER export results in redistribution of all the Golgi residents examined so far into the ER, which suggests that the Golgi functions in dynamic equilibrium with the ER, rather than as a stable entity [13]. Importantly, reversal of the ER export block enables the cell to generate a subcompartmentalized Golgi apparatus de novo [14]. This process may be conceived of as proceeding in discrete steps with separate molecular requirements. First, Golgi proteins are concentrated and exported out of the ER by COPII vesicles, which form pleiomorphic membrane structures termed vesicular tubular clusters (VTCs), also known as the ER Golgi intermediate compartment. Second, subcompartments are generated from the noncompartmentalized VTC, as evidenced by segregation of cis, medial and trans Golgi proteins. Third, these compartmentalized structures are then transported along microtubules to the cell center, where, in the fourth step, they are linked to form a single perinuclear Golgi ribbon. As the first two steps directly impact subcompartmentalization, we will focus on the mechanisms that mediate these reactions.

Generation of Golgi subcompartmentalization by maturation and local recycling During de novo Golgi biogenesis, COPII vesicles carry out the initial concentration and export of Golgi proteins from the ER. Repeated rounds of COPII vesicle fusion generate VTCs, which further concentrate Golgi proteins by recycling escaped ER proteins back to the ER [15,16]. Compartmentalization might start with sorting of cis Golgi proteins away from the others by COPI vesicles. These vesicles fuse with newly generated COPII vesicles or VTCs, thus enriching them in cis elements. Next, the medial Golgi proteins are sorted into vesicles, which fuse with the cis-enriched VTC, thus allowing it to mature. As shown in Figure 1, such iterative local recycling of residents, induced by the COPI coat having a high affinity for early Golgi proteins and decreasing affinities for later components, could initiate compartmentalization [16,17,18]. Recent morphological evidence suggests that ER export might occur even in zones devoid of COPII vesicles, through tubular outgrowths that differentiate into maturing VTCs [19]. www.sciencedirect.com

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De novo generation of Golgi subcompartments by iterative local recycling. The model assumes that cis enzymes are the most efficiently recycled while trans enzymes are the least. Local recycling, as opposed to recycling via the ER, allows maintenance of Golgi subcompartments [17].

Compartmentalization driven by maturation may further be aided by the biased affinities of COPII for different Golgi proteins. Early (cis) Golgi proteins seem to be the first to be exported, and the initial VTCs generated are enriched in these [20,14]. This suggests that the cis compartments are generated early during biogenesis, followed by the later compartments. Consistent with this, segregation between the cis Golgi protein GM130 and the medial Golgi enzyme Mannosidase-II is observed very early during Golgi biogenesis [14]. Continuous maturation and local recycling might also maintain subcompartmentalization at steady state. Indeed, as VTCs will be de-enriched for Golgi components, the process is likely to be much more efficient. Further, the COPII bias towards cis-Golgi residents would ensure that the input to the Golgi from the ER would be enriched in early components, which favors subcompartmentalization.

Mechanisms of local recycling How is the fidelity of local recycling maintained? One possibility is that different coats have different affinities for various SNAREs [10]. Diversity in cargo specificity of both COPII and COPI coats might be achieved by their existence as multiple distinct heteromers, differing in their cargo-binding subunits. Consistent with this, mammalian cells have at least four isoforms of the sec24 subunit of COPII [21]. Also, the proteins Lst1p and Sfb2p have been shown to substitute for Sec24p in yeast, to form COPII vesicles with different cargo specificities [22–24]. Similar isoform substitutions have also been postulated in COPI coat complex formation [25]. When coordinated, the differential sorting achieved by such heteromers could serve to concentrate specific SNAREs in different compartments, and to generate gradients of SNARE concentrations along the secretory pathway. This in turn will dictate specificity of protein transfer across compartwww.sciencedirect.com

ments and help maintain a subcompartmentalized Golgi (Figure 2). As predicted by the model, at least three specific sets of SNAREs act in traffic between the ER and the Golgi. In all cases, the t-SNARE is composed of three separate proteins, which provide three of the four helices. Syntaxin5, when complexed with sec22 and membrin, forms a t-SNARE that interacts specifically with the v-SNARE bet1. When complexed with GOS28 and Ykt6, Syntaxin5 forms a t-SNARE that binds GS15 [26,27]. These two v–tSNARE sets might mediate fusion of COPII to form VTCs, and fusion of locally recycling COPI vesicles, respectively [27,28]. Retrograde vesicles that fuse with the ER are thought to contain the v-SNARE Sec20, which binds the t-SNARE complex of Ufe1, Sec22 and Slt1 [29]. Careful analyses of the sorting of these SNAREs by different coats will give us an insight into the validity of the SNARE gradient model. In the Golgi, local recycling may be enhanced by retention of vesicles in the local environment by an additional layer of protein interactions, which tether these vesicles to the acceptor compartment. As the proposed lengths of such vesicle ‘tethers’ are more than the distance between the cisternae, it is possible that tethering is established even before vesicle budding is complete. Tethers might thus allow directional intercisternal transport using a single set of SNAREs. Two classes of proteins have been proposed to form COPI vesicle tethers. The first belong to the golgin family of long coiled-coil proteins. Protein-interaction and vesicledocking studies carried out in vitro suggest that giantin, which is present on the vesicle, might be linked to membrane-associated GM130 by the docking factor p115, and that formation of this tether is a prerequisite for fusion [30–32]. Recently, it was also observed that Current Opinion in Cell Biology 2005, 17:369–375

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Figure 2

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Hypothetical scheme for compartmentalization based on different affinities of coats for various SNARES and guanine nucleotide exchange factors (GEFs), resulting in distinct concentration gradients of proteins. Each compartment (ER, cis, trans, PM) is marked by an enriched GEF (labeled G1–G5), and an enriched t-SNARE (T1–T5). The GEF determines coat formation by a corresponding coat (C1–C5), which mediates the sorting of specific v-SNAREs (V1–V5) into vesicles. Cognate t-SNAREs define the acceptor compartment for vesicles. Homotypic fusion, recycling and homeostatic mechanisms, defined by the same rules, allow, in principle, the de novo generation of the cis and trans compartments from the ER and PM. This requires that the coats sequester SNAREs on compartments and activate specific SNAREs for fusion during vesicle formation. Concentration gradients of the components that will be generated by such a system are indicated at the top of the figure.

vesicles containing golgin-84 are retained on slides coated with the giantin-related protein CASP [33]. On the basis of the relative enrichment of Golgi enzymes and relative depletion of a forward cargo marker in CASP-binding vesicles, as opposed to p115-binding vesicles, it was proposed that distinct tethers might dictate directionality of COPI vesicles. Interestingly, both giantin and golgin84 were present in both classes of vesicles. Hence, if such tethers do define fusion targets, additional mechanisms must selectively activate/inactivate one vesicle-associated tether component. A major shortcoming of the golgin-based tether model is that in vivo evidence strongly argues against a required role for either of these tethers in vesicle fusion and Golgi subcompartmentalization. Depletion of giantin, GM130 or golgin-84 in intact cells causes neither abundant accumulation of Golgi-derived vesicles nor collapse of Golgi subcompartments [34–36]. Significantly, p115 is required for vesicle fusion and Golgi subcompartmentalization Current Opinion in Cell Biology 2005, 17:369–375

[34,37]. However, the mechanism of action of p115 involves its SNARE-binding domain and not its giantinor GM130-binding domains [37]. Thus, it currently appears that p115 may be a SNARE regulator rather than a vesicle tether. On the other hand, in vivo data support the involvement of large multiprotein complexes in tethering Golgi vesicles [38]. Depletion of COG3, a subunit of the COG complex that interacts with both COPI and Golgi SNAREs, causes apparent accumulation of Golgi-derived vesicles containing specific proteins including v-SNAREs [39]. Similarly, in yeast, the TRAPP complex, which directly binds COPII vesicles, might tether them to the Golgi apparatus [40]. The initial stages of de novo Golgi biogenesis present several problems for local recycling. A newly budded vesicle containing an active v-SNARE has many potential fusion sites on the ER. What prevents such futile backwww.sciencedirect.com

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fusion of vesicles? Also, as the compartments themselves have active SNAREs, what prevents them from fusing with each other? Similar questions apply to steady-state vesicle transport, as SNAREs have to be recycled back for multiple rounds of fusion. An obvious solution is to keep the SNAREs inactive until a specific fusion reaction is called for. A membrane-proximal ‘switch’ that needs to be flipped to make them fusion-competent has been identified on t-SNAREs [41,42]. Post-translational modifications such as phosphorylation, and additional proteins such as GATE-16 and LMA1, which are transferred onto free SNAREs by NSF [43,44], also might keep SNAREs from participating in unwanted fusion. A specific role in preventing back-fusion of COPII vesicles with the ER has been attributed to Tip20, a SNARE-like protein, which interacts with the retrograde ER SNARE complex [45]. Interestingly, the presence of a fifth SNARE can preclude formation of fusogenic SNARE complexes [46]. It is possible that recycling SNAREs act in this way as inhibitory molecules or i-SNAREs, which prevent non-specific fusion. These may later be sequestered away by target-associated proteins to allow specific fusion. Strikingly, cis Golgi SNAREs inhibit the fusion mediated by trans Golgi SNAREs, and vice versa. Such automatic generation of complementary gradients of i-SNAREs ensures gradients of fusogenic activity of SNAREs, beyond the activity gradients resulting from their concentrations. As uncoating of a vesicle is a prerequisite for fusion, it might be the case that coats sequester SNAREs and inhibit wanton vesicle fusion. Such sequestration, if it happens on compartments, might be the mechanism that prevents their indiscriminate fusion with each other. Consistent with this, COPI disassembly is much slower on flatter membranes, as opposed to vesicles [47]. In addition, coat proteins themselves might also activate specific v-SNAREs and inactivate all other recycling SNAREs during vesicle formation, thus ensuring only one potential SNARE pair per vesicle (Figure 2).

Comparison to post-mitotic Golgi reassembly Most evidence suggests that during mitosis, the mammalian Golgi apparatus is converted into an abundant population of vesicles that are dispersed in the metaphase cell. After cytokinesis, each of the daughter cells then reassembles a compartmentalized Golgi apparatus. One scenario is that during mitosis, the vesicle fusion machinery in the Golgi is inhibited, while vesicle formation continues. It is noteworthy, however, that required roles in mitotic Golgi disassembly for either tether/SNARE inhibition or COPI function still remain to be demonstrated [48]. If so, these reactions would convert the Golgi stack into distinct sets of vesicles that retain their interphase subcompartmental enrichment and programming for targeted fusion. Thus, during post-mitotic reassembly, vesiwww.sciencedirect.com

cles that were originally derived from the cis Golgi, and which contain cis Golgi residents, fuse with newly generated VTCs, thereby driving maturation of the VTCs. Further maturation by fusion of the remaining vesicles and the initiation of local recycling would then establish later subcompartments. According to this view, postmitotic Golgi assembly essentially involves the same steps and mechanisms as steady-state maintenance of the Golgi and de novo Golgi biogenesis from the ER. As the initial distribution of Golgi components in the ER, vesicles and Golgi elements differ significantly in these three states, robust homeostatic mechanisms are implicit in establishing proper Golgi organization from distinct starting points.

Conclusions Efficient incorporation of newly synthesized or cycling Golgi proteins during ER exit, followed by active sorting of earlier components into retrograde vesicles to allow iterative local recycling, might suffice to create and maintain a subcompartmentalized Golgi apparatus. The accuracy of local recycling might be achieved by SNARE gradients generated by differential affinities of coats, with additional fine control provided by SNARE-interacting proteins that regulate aspects of the SNARE association– dissociation cycle. Further elucidation of the interplay between coats and SNAREs will help us establish the minimal system required to generate subcompartments in the Golgi.

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Golgi and back. They show that in the absence of Tip20, COPII vesicles never reach the Golgi, which might be due to their increased consumption by back-fusion with the ER. 46. Varlamov O, Volchuk A, Rahimian V, Doege CA, Paumet F,  Eng WS, Arango N, Parlati F, Ravazzola M, Orci L et al.: i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion. J Cell Biol 2004, 164:79-88. Vesicle fusion mediated by fusogenic SNAREs in vitro is inhibited by the addition of a fifth SNARE that does not belong in the complex. As cisGolgi SNAREs inhibit trans-Golgi SNARE-pairs, and vice versa, the SNARE gradients present across the Golgi stacks might be sharpened by automatic generation of complementary gradients of i-SNAREs. 47. Bigay J, Gounon P, Robineau S, Antonny B: Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature. Nature 2003, 426:563-566. 48. Puthenveedu MA, Linstedt AD: In search of an essential step during mitotic Golgi disassembly and inheritance. Exp Cell Res 2001, 271:22-27.

Current Opinion in Cell Biology 2005, 17:369–375