Transport and sorting in the Golgi complex: multiple mechanisms sort diverse cargo

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ScienceDirect Transport and sorting in the Golgi complex: multiple mechanisms sort diverse cargo Gaelle Boncompain1 and Aubrey V Weigel2 At the center of the secretory pathway, the Golgi complex ensures correct processing and sorting of cargos toward their final destination. Cargos are diverse in topology, function and destination. A remarkable feature of the Golgi complex is its ability to sort and process these diverse cargos destined for secretion, the cell surface, the lysosome, or retained within the secretory pathway. Just as these cargos are diverse so also are their sorting requirements and thus, their trafficking route. There is no one-size-fits-all sorting scheme in the Golgi. We propose a coexistence of models to reconcile these diverse needs. We review examples of differential sorting mediated by proteins and lipids. Additionally, we highlight recent technological developments that have potential to uncover new modes of transport. Addresses 1 Institut Curie, PSL Research University, CNRS UMR144, F-75005 Paris, France 2 Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, USA Corresponding author: Boncompain, Gaelle ([email protected])

Current Opinion in Cell Biology 2018, 50:94–101 This review comes from a themed issue on Cell architecture Edited by Celeste Nelson and Franck Perez

https://doi.org/10.1016/j.ceb.2018.03.002 0955-0674/ã 2018 Elsevier Ltd. All rights reserved.

[1]. Many reviews have addressed the details of these different models [2–4]. The most popular models include cisternal maturation [5], vesicular transport between stable compartments [6] and the rapid partitioning model [7]. Each of these models possesses strengths and weaknesses, and on their own does not fully accommodate all of the data present in the literature. Consequently, this has left the field ambivalent, making it arduous to declare one single model as the end-all method of transport across this organelle. Cargo proteins vary considerably in size, membrane association, destination and modifications, thus, each has its own specialized sorting and trafficking requirements. The Golgi complex is a dynamic organelle and has to handle the diversity of secretory cargos and to ensure their correct sorting and targeting all while maintaining its own identity. Cargos are sorted and segregated to be excluded from or packaged into transport intermediates. While there are likely not as many different sorting rules as there are cargo, we suggest that different sorting rules may apply to different classes of cargo. In this minireview, we discuss mechanisms of anterograde transport of cargos through the Golgi apparatus in mammalian, non-polarized cells. We propose that different sorting mechanisms coincide to ensure trafficking of diverse cargos and discuss the regulatory roles of both proteins and lipids. In addition, we highlight emerging technologies that may illuminate previously unappreciated differences in Golgi complex dependent sorting and trafficking.

Diversity of cargos Introduction The secretory pathway is an essential process for eukaryotic cells. It is responsible for the synthesis, processing and delivery of a diverse collection of proteins to their final destination. The journey begins in the endoplasmic reticulum (ER) where proteins are synthesized and translocated. Cargo proteins are then transported to the Golgi complex, a polarized organelle composed of stacked cisternae, where cargo is eventually, processed and sorted. Finally, cargo is exported to its destination compartment, such as the plasma membrane or lysosomes, while resident Golgi proteins remain. The mechanisms for cargo transport through the Golgi complex have been the subject of a long-lasting debate Current Opinion in Cell Biology 2018, 50:94–101

The variety of cargos that transit the secretory system is vast. In mammalian cells, about 30% of newly synthetized proteins enter the secretory pathway. There is substantial heterogeneity in size, topology and function. A secretory cargo can be a soluble protein, a transmembrane protein or even anchored to a lipid within the membrane (Figure 1a). Soluble cargo can range from less than 10 kDa, such as some cytokines, all the way up to 100s of kDa (Human monomeric type I Collagen is 400 kDa in its trimeric form). Similarly, transmembrane proteins vary greatly in size and shape. On one end of the spectrum is a simple, single-pass transmembrane protein. While at the other end lies a multi-pass protein, that assembles several subunits together, such is the case for many ion and ligand gated channels. Mixed into this assortment of secretory cargos is lipid anchored proteins, which includes prenylated proteins, fatty acylated proteins and www.sciencedirect.com

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Diversity of the secretory pathway. (a) Cargos entering the secretory pathway are diverse, displaying a large variation in size and topology. By definition, at least part of the protein is contained within the lumen of the secretory compartments. This includes small soluble proteins such as cytokines, large soluble proteins such as collagens, GPI-anchored proteins, transmembrane proteins bearing either one or several transmembrane domains. (b) The secretory pathway is not composed of a single route from the endoplasmic reticulum to the plasma membrane, but rather the intersection of several routes. The destination compartments of cargos are diverse with cargos addressed to the plasma membrane, to the endolysosomal compartments or retained within Golgi apparatus. Consequently, their route through the Golgi is similarly diverse.

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glycosylphosphatidylinositol-linked (GPI) proteins. Furthermore, maturation of glycoproteins by resident enzymes takes place within the Golgi complex. To reach their end destination, the heterogeneous population has to be sorted; cargo proteins each move to the appropriate destination while Golgi resident proteins remain. The population is not static, rather, Golgi cargo composition can shift dramatically to respond to cell needs. In essence, the secretory pathway is not a single route but rather several routes that diverge within the Golgi apparatus (Figure 1b).

Intra-Golgi trafficking: a summary of observations and evaluation of models How does this diverse array of cargos traffic across the Golgi? In order to address this question a host of observations needs to be taken into consideration. We highlight here some essential studies and key points that should be acknowledged by any proposed model, or combination thereof, for Golgi traffic in mammalian cells. First, The Golgi complex is composed of distinct compartments, polarizing the organelle cis to trans based on glycosylation enzyme composition within each compartment [8,9]. Second, These resident Golgi enzymes can quickly move between compartments [10]. Third, Cargo proteins, both large and small, can move across the mammalian Golgi stacks in a vectoral fashion cis-to-trans [11–14]. Fourth, Cargo proteins can traverse the Golgi at very different rates [7,15,16]. Fifth, Cargo molecules exit the Golgi complex with exponential kinetics [7]. For several years, the field has been predominately arguing between two models of cargo progression through the Golgi: cisternal maturation versus vesicular transport, which have been extensively described in the past [3,5,17–19] (Box 1). Briefly, the cisternal maturation model views cisternae as transient carriers born out of the ER and maturing over time into a TGN cisterna in a sort of conveyor belt fashion. Conversely, in the vesicular transport model the Golgi is comprised of stable compartments, each of which contain a unique set of resident Golgi enzymes. Cargo is transported from compartment to compartment in an assembly-line manner via vesicular trafficking. For more than a decade now, accumulating data has oriented the field toward the cisternal maturation model and away from vesicular trafficking [3]. While cisternal maturation can readily explain the existence of discrete compartments, the polarized distribution of resident Golgi enzymes and transport of large secretory cargo, it fails to explain the variable trafficking rates of different secretory cargos and mono-exponential export kinetics from the Golgi complex. Instead this model would predict linear export kinetics. The observation of mono-exponential kinetics birthed a third, alternative model, namely the rapid-partitioning Current Opinion in Cell Biology 2018, 50:94–101

Box 1 Maturing versus stable cisternae: clues obtained by controlled aggregation. Two studies using controlled aggregation of proteins were conducted to revisit the question of Golgi cisternae maturation. Rizzo, Parashuraman et al. used reversible aggregation of a Golgi resident enzyme (mouse a-1,2-mannosidase IB, MANI) [63]. They demonstrated that MANI aggregates were not able to enter into the carriers and moved forward in the Golgi stack, favoring the maturation model. However, in this study, only the retrograde movement of resident enzymes was analysed, no anterograde secretory cargo was used. Lavieu et al. engineered protein ‘staples’ using the aggregation of a single pass transmembrane cargo (CD8) or a soluble cargo (hGH) combined with a 16  C temperature block [64]. Their observations were contradictory to the cisternal maturation model predictions. They observed that these protein ‘staple’ aggregates were not able to move forward in the Golgi stack, while soluble aggregates did move forward. Interestingly, soluble and membrane aggregates were segregated within the cisternae, with soluble aggregates being localized to the rims of cisternae. From these results, Lavieu et al. proposed that only rims of cisternae are mobile. The discrepancies between these two studies likely originates in the method of aggregation itself and in the artificial tools and non-physiological conditions used.

model [7]. Here, the Golgi exists as a single interconnected compartment that contains processing and export domains, based on a lipid gradient. Cargo arrives from the ER, quickly partitions between the two domains, then stochastically exits from every level of the Golgi to their final destination. The residence time in processing domains, the diffusion properties of the cargo and the site of Golgi exit explain observations of different export kinetics from different cargos. An additional consideration is intercisternal connections [20,21]. Beznoussenko, Parashuraman et al. elegantly demonstrated that small soluble cargos such as albumin and antitrypsin were present in the intercisternal connections whereas large cargos (procollagens) are excluded [15]. Small cargos traverse the Golgi stack faster than procollagens. Their results indicate that transport of small soluble cargos occur via the intercisternal continuities enabling faster transport compared to collagens which progress at the cisternal maturation rate. This study provided evidence of multiplicity of the mechanisms of transport through the Golgi. Importantly, and a central theme to this review, the described mechanisms of Golgi-dependent transport do not have to be exclusive [22] (Figure 2). The Golgi complex might handle trafficking in a cargo-dependent way and adapt transport mechanisms to the characteristics of the secretory cargo. Otherwise, it would be difficult to envisage a single mode of transport of such diverse cargos and reconcile accumulating data. Large cargos such as collagens will move forward thanks to maturation of cisternae in which they are confined. Tubular connections across cisternae will allow small cargos (either soluble or transmembrane) to traverse the Golgi faster than www.sciencedirect.com

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Mechanisms of intra-Golgi transport of cargos. Several non-exclusive mechanisms of cargo transport through the Golgi complex exist. Left: rapid partitioning model proposed by Patterson et al. According to this model, cargos are segregated in processing and export domains. The partitioning is based on biophysical properties of cargos and on the gradient of lipids across the Golgi. In the processing domains, cargos are modified by the resident Golgi proteins such as the glycosylation enzymes while export domains present in all cisternae allows exit of cargos. Middle: cisternal maturation and transport kinetics influenced by diffusion properties of the cargos. Membranes containing cargos move forward and mature by successive acquisition of resident Golgi proteins. The maturing cisternae advance as a conveyor belt for cargos. Ultimately at the level of the trans Golgi, membranes breakdown and post-Golgi carriers are formed to mediate the transport of cargos. Diffusion properties of cargos through the interconnected cisternae might explain the different kinetics of transport through the Golgi. Slow diffusing cargo will advance at the speed of a maturing cisterna whereas highly diffusive proteins will reach export domains quickly. Right: transport of soluble cargos via intercisternal continuities and of large aggregates by cisternal maturation as demonstrated by Beznoussenko, Parashuraman et al. Cisternae are connected by continuities. Small soluble cargos such as albumin were observed in the continuities speeding up their transport across the stack. Large cargos such as collagens are excluded from the continuities. Consequently, large cargos advance slower thanks to the cisternal maturation.

the maturation speed. In addition, diffusion properties of the cargos will influence their kinetics of intra-Golgi transport. Slow diffusing cargo will advance at the speed of a maturing cisterna whereas highly diffusive proteins will reach export domains quickly. Intra-Golgi retrograde flow will recycle back the resident Golgi enzymes and membranes to ensure homeostasis of the Golgi complex. Furthermore, discrepancies in lipid composition www.sciencedirect.com

throughout the secretory pathway aid in the partitioning and sorting of cargos.

Protein and lipid mediators of differential trafficking Once cargo has been processed and sorted it leaves the Golgi predominantly at the level of the trans-Golgi network (TGN) in post-Golgi carriers. Historically this Current Opinion in Cell Biology 2018, 50:94–101

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sorting was thought to occur only in the TGN [23]. However, studies in both polarized [24] and non-polarized cells have shown that cargos can be sorted at the level of the main Golgi complex before reaching the TGN [15,16,25]. There are several protein mediators of differential Golgi trafficking, including SNARE proteins, golgins, Rab GTPases, adaptor protein complexes (APs) and Golgiassociated, g-adaptin homologous, ARF-interacting proteins (GGAs). SNARE proteins, while regulators of global trafficking, are limited at the Golgi [26]. The family of golgins is composed of large coil-coiled proteins present at the cytoplasmic face of the Golgi [27] and have been shown by several studies to be involved in the differential transport of cargos. For instance, golgin-97 regulates the transport of E-cadherin but is not involved in the transport of TNF [28]. Contrariwise, TNF transport is dependent on golgin-245, the latter having no effect on the trafficking of E-cadherin [29]. Hicks et al. demonstrated that golgin-160 is required for the transport of beta1-adrenergic receptor, but not VSVG, to the cell surface [30]. A recent study provided elegant evidence that golgins specifically tether distinct transport vesicles at Golgi membranes [31]. Using synchronized transport out of the ER of a secreted protein, they showed that GMAP-210 and GM130 were able to capture ER derived vesicles. They also demonstrated that TMF and golgin84 captured vesicles containing the resident Golgi enzyme GalNAc-T2. Although this work illustrated the selectivity of golgins regarding the origin of transport vesicles, further work using different cargos would be required to assess the capacity of golgins to capture diverse cargos in a specific way. The molecular determinants regulating the specificity of golgins are unknown. However, GMAP-210 has recently been shown to recognize specific lipids to tether transport vesicles at the cisface of the Golgi [32] providing valuable insight into how golgins might filter transport vesicles, albeit in a noncargo-specific manner. Rab GTPases recruit various effectors, especially molecular motors, to ensure correct transport of a variety of cargos [33]. Among the 60 Rab GTPases, Rab6, Rab19, Rab33, Rab34, Rab36 and Rab39 are primarily localized at the Golgi complex. Rab6 is the most abundant Rab GTPase at Golgi membranes [34]. It regulates the size of the Golgi apparatus, plays a pivotal role in secreting cargos [35,36] and was recently shown to recruit MyosinII and KIF20A to affirm fission of transport vesicles [37,38]. Rab43 has been shown to regulate sorting of adrenergic receptors [39] and of AE1-4 anion exchanger [40] but not VSVG. APs and GGAs take over cargos at the level of the TGN [41]. AP-1, AP-3, AP-4 and the three GGAs are enriched Current Opinion in Cell Biology 2018, 50:94–101

in TGN membranes and are involved in transport from TGN to the endolysosomal compartments. The adaptors recognize sorting signals in the cytoplasmic tail of secretory proteins and recruit coat protein (e.g. clathrin). The tyrosine-based signal YXXF is recognized by APs [42] as well as the di-leucine-based signal (D/E)XXL(L/I) [43] whereas DXXLL is recognized by GGAs [44]. A recent study showed that segregation of cargos destined to the endolysosomal compartments, namely LAMP-1, TfR and CD-MPR, occurs early in the Golgi complex and independently of the detection of the cytoplasmic signals by APs [25]. Another contributor to cargo sorting arises from the membrane’s lipid composition. Three major types of lipids exist in secretory system membranes: glycerophospholipids, sphingolipids, and sterols such as cholesterol [45]. Cholesterol and glycerophospholipids are synthesized in the ER, while the production of sphingolipids is housed within the Golgi. The lack of cholesterol and sphingolipid in the ER makes the membrane readily deformable, thinner and consequently much easier for newly synthesized proteins to be inserted into the bilayer and continue folding. The gradient of lipids presented across the Golgi is the basis for models such as the rapid partitioning model. For instance, plasma membrane bound cargo naturally partitions into domains where the membrane is thicker due to the elevated concentration of sphingomyelin and cholesterol. This model is supported by evidence that proteins are retained or exported from the Golgi with dependency on their membrane length [46]. Phosphatidylinositol 4-phosphate (PI4P) is another key lipid in the secretory pathway. PI4P populates the Golgi complex where it is produced, the plasma membrane as well as late endosomes [47] and is required in multiple cellular functions including vesicular traffic [48]. Several TGN-associated proteins have been found to bind PI4P including an AP-1 accessory protein, epsinR [49], AP-1 [50], FAPP1, FAPP2, OSBP [51,52], and CERT [53]. More recent work has revealed an additional role of PI4P, linking the lipid exchange cycles of lipid transfer proteins, such as OSBP, in a mutually exclusive coupling to cholesterol [54,55]. Just as there may not be a single model that exists for all cargo to transit the Golgi, there may also exist several models that explain its exit from the Golgi. We propose that the route that cargo takes through the Golgi and to its final compartment may be governed by the cargo, rather than a global model that all cargos must neatly fit within.

Tools to dissect Golgi-dependent transport With the advent of new technology comes the ability to answer outstanding questions, and revisit already answered questions with a new perspective. Here we www.sciencedirect.com

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summarize classic tools used to study the secretory pathway and highlight emerging technologies that may help differentiate previously unappreciated differences in Golgi complex sorting and trafficking. The visualization of secretory proteins in real time was powered up by the discovery of green fluorescent protein (GFP) and its spectral variants [56]. However, membrane trafficking is balanced by anterograde and retrograde fluxes of cargos, making it difficult to see the trafficking of single cargos amongst the sea of other cargos proteins and resident enzymes. As a solution, several tools have been developed to study the synchronized release of cargo protein from the ER.

Conclusion Undoubtedly, the Golgi complex is the major secretory station for membrane trafficking. Several intersecting transport routes from diverse cargos traverse this organelle. The mechanisms underlying the diverse routes are not fully understood. Studies assessing these questions have become rarer during the last decade. However, work is still needed to increase and consolidate our understanding of Golgi-dependent transport. We encourage the field to revisit past results with the new perspective of recent technological advancements in hopes of revealing insights into the multiple paths through the Golgi that effectively deliver diverse cargo to their destinations.

Acknowledgements One such classical tool is the thermosensitive glycoprotein of vesicular stomatitis virus (VSVGtsO45). At restrictive temperature (39.5  C) VSVGtsO45 is not able to oligomerize, which is a prerequisite for its exit from the ER [57]. Shifting to a permissive temperature (32  C) allows oligomerization of VSVGtsO45, which then enters the secretory pathway [58]. This tool, developed in the 90s, is powerful and has brought an enormous understanding of secretory traffic to the field. The retention using selective hooks (RUSH) assay was engineered by the Perez lab [16], allowing synchronization of a cargo of interest at physiological conditions for mammalian cells. The cargo of interest is retained in the ER because of its fusion to a streptavidin binding peptide (SBP), which interacts with streptavidin fused to an ER hook. Interaction is then released by addition of biotin and the cargo traffics in a synchronous way via the Golgi toward its destination compartment. Similar work has been done using a small ligand [59] or light [60] to aggregate and synchronously release cargo from the ER. As mentioned above, fusion of the cargo of interest with fluorescent proteins, and ideally in combination with a synchronization assay, enables monitoring a wave of protein transport. This is especially useful to study segregation and sorting mechanisms of cargos at the level of the Golgi complex. Combining low phototoxicity and low photobleaching, lattice light sheet microscopy allows imaging of whole cell volumes at high spatio-temporal resolution [61], improving upon conventional confocal microscopy. It has the potential to reveal incredible spatio-temporal details of membrane trafficking. In order to assess cargo partitioning along the secretory pathway, especially at the level of the Golgi complex, a broad color palette of fluorescent proteins and dyes in combination with versatile tools needs to be employed to monitor the simultaneous transport of several cargos within the same cell. For instance, following work by Valm and Cohen et al. [62], a live-cell, multispectral approach could be applied to investigate the transit of several cargos through the secretory system and allow the monitoring of their interactions with each other and other resident organelle proteins. www.sciencedirect.com

We acknowledge Dr. Carolyn Ott for careful reading of the manuscript. G. Boncompain is working in the lab of F. Perez. Work in F. Perez’s lab is funded by Centre National de la Recherche Scientifique (CNRS), Agence Nationale pour la Recherche (ANR) et la Fondation pour la Recherche Me´dicale (FRM). A.V. Weigel is working in the lab of J. LippincottSchwartz. Work in the lab of J. Lippincott-Schwartz lab is funded by Howard Hughes Medical Institute (HHMI). Collaborative work between G. Boncompain and A.V. Weigel was funded by the Labex CellTisPhyBio and by the visitor program of HHMI — Janelia Research Campus.

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Current Opinion in Cell Biology 2018, 50:94–101