Signaling Circuits on the Golgi Complex

Numerous components of signaling pathways involved in key cellular processes reside on the Golgi complex. Here, we will focus on the roles of signaling proteins that regulate cargo trafficking along the anterograde and retrograde pathways. Emphasis will also be put on the effects of these regulatory proteins on the maintenance of the structure and function of the Golgi, and in particular on the phosphorylation of key components of the transport machinery. These pathways position the Golgi complex as a central hub in the regulation of cell signaling. To date, however, the activation and coordination of these signaling molecules remain a mystery. Being able to describe the interplay between several of these signaling pathways and secretion, and the flow of information through these pathways, will help us to understand how the secretory machinery works and how it interacts with other cellular functions. This will also advance our understanding of how the secretory pathway functions under physiological circumstances, and how its dysregulation can initiate pathological conditions.     

Re'COG'nition at the Golgi

The conserved oligomeric Golgi (COG) complex co-ordinates retrograde vesicle transport within the Golgi. These vesicles maintain the distribution of glycosylation enzymes between the Golgi's cisternae, and therefore COG is intimately involved in glycosylation homeostasis. Recent years have greatly enhanced our knowledge of COG's composition, protein interactions, cellular function and most recently also its structure. The emergence of COG-dependent human glycosylation disorders gives particular relevance to these advances. The structural data have firmly placed COG in the family of multi-subunit tethering complexes that it shares with the exocyst, Dsl1 and Golgi-associated retrograde protein (GARP) complexes. Here, we review our knowledge of COG's involvement in vesicle tethering at the Golgi. In particular, we consider what this knowledge may add to our molecular understanding of vesicle tethering and how it impacts on the fine tuning of Golgi function, most notably glycosylation.     

A Golgi Lipid Signaling Pathway Controls Apical Golgi Distribution and Cell Polarity during Neurogenesis

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Actin acting at the Golgi

The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in trafficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers.     

Metabolic Activation of the HOG MAP Kinase Pathway by Snf1/AMPK Regulates Lipid Signaling at the Golgi

Phosphatidylinositol‐4‐phosphate (PI(4)P) is an important regulator of Golgi function. Metabolic regulation of Golgi PI(4)P requires the lipid phosphatase Sac1 that translocates between endoplasmic reticulum (ER) and Golgi membranes. Localization of Sac1 responds to changes in glucose levels, yet the upstream signaling pathways that regulate Sac1 traffic are unknown. Here, we report that mitogen‐activated protein kinase (MAPK) Hog1 transmits glucose signals to the Golgi and regulates localization of Sac1. We find that Hog1 is rapidly activated by both glucose starvation and glucose stimulation, which is independent of the well‐characterized response to osmotic stress but requires the upstream element Ssk1 and is controlled by Snf1, the yeast homolog of AMP‐activated kinase (AMPK). Elimination of either Hog1 or Snf1 slows glucose‐induced translocation of Sac1 lipid phosphatase from the Golgi to the ER and thus delays PI(4)P accumulation at the Golgi. We conclude that a novel cross‐talk between the HOG pathway and Snf1/AMPK is required for the metabolic control of lipid signaling at the Golgi.     

Sphingomyelin-enriched Microdomains at the Golgi Complex

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the alpha- and beta-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.     

Protein quality control at the Golgi

The majority of the proteome in eukaryotic cells is targeted to organelles. To maintain protein homeostasis (proteostasis), distinct protein quality control (PQC) machineries operate on organelles, where they detect misfolded proteins, orphaned and mis-localized proteins and selectively target these proteins into different ubiquitin-dependent or -independent degradation pathways. Thereby, PQC prevents proteotoxic effects that would disrupt organelle integrity and cause cellular damage that leads to diseases. Here, we will discuss emerging mechanisms for PQC machineries at the Golgi apparatus, the central station for the sorting and the modification of proteins that traffic to the endo-lysosomal system, or along the secretory pathway to the PM and to the extracellular space. We will focus on Golgi PQC pathways that (1) retrieve misfolded and orphaned proteins from the Golgi back to the endoplasmic reticulum, (2) extract these proteins from Golgi membranes for proteasomal degradation, (3) or selectively target these proteins to lysosomes for degradation.     

Entry at the trans-Face of the Golgi

The trans-Golgi network (TGN) receives a select set of proteins from the endocytic pathway—about 5% of total plasma membrane glycoproteins (Duncan and Kornfeld 1988). Proteins that are delivered include mannose 6-phosphate receptors (MPRs), TGN46, sortilin, and various toxins that hitchhike a ride backward through the secretory pathway to intoxicate cells after they exit into the cytoplasm from the endoplasmic reticulum (ER). This article will review work on the molecular players that drive protein transport from the endocytic pathway to the TGN. Distinct requirements have revealed multiple routes for retrograde transport; in addition, the existence of multiple, potential coat proteins and/or cargo adaptors imply that multiple vesicular transfers are likely involved. Several comprehensive reviews have appeared recently and should be sought for additional details (Bonifacino and Rojas 2006; Johannes and Popoff 2008).     

The Golgi apparatus at the cell centre

In non-polarised mammalian cells, the Golgi apparatus is localised around the centrosome and actively maintained there. Microtubules and molecular motor activity are required for determining both the localisation and organisation of the Golgi apparatus. Other factors, however, also appear necessary for regulating both the static steady-state distribution of this organelle and its relationship with microtubule minus-end-anchoring activities of the centrosome. Several non-motor microtubule-binding proteins have now been found to be associated with the Golgi apparatus. Recent advances suggest that, in addition to important roles in cell motility, polarisation and differentiation, the interplay between Golgi apparatus and centrosome could participate in other physiological processes such as intracellular signalling, mitosis and apoptosis.     

T Cell Receptor Signaling Co-regulates Multiple Golgi Genes to Enhance N-Glycan Branching

T cell receptor (TCR) signaling enhances β1,6GlcNAc-branching in N-glycans, a phenotype that promotes growth arrest and inhibits autoimmunity by increasing surface retention of cytotoxic T lymphocyte antigen-4 (CTLA-4) via interactions with galectins. N-Acetylglucosaminyltransferase V (MGAT5) mediates β1,6GlcNAc-branching by transferring N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to N-glycan substrates produced by the sequential action of Golgi α1,2-mannosidase I (MIa,b,c), MGAT1, α1,2-mannosidase II (MII, IIx), and MGAT2. Here we report that TCR signaling enhances mRNA levels of MIa,b,c and MII,IIx in parallel with MGAT5, whereas limiting levels of MGAT1 and MGAT2. Blocking the increase in MI or MII enzyme activity induced by TCR signaling with deoxymannojirimycin or swainsonine, respectively, limits β1,6GlcNAc-branching, suggesting that enhanced MI and MII activity are both required for this phenotype. MGAT1 and MGAT2 have an ∼250- and ∼20-fold higher affinity for UDP-GlcNAc than MGAT5, respectively, and increasing MGAT1 expression paradoxically inhibits β1,6GlcNAc branching by limiting UDP-GlcNAc supply to MGAT5, suggesting that restricted changes in MGAT1 and MGAT2 mRNA levels in TCR-stimulated cells serves to enhance availability of UDP-GlcNAc to MGAT5. Together, these data suggest that TCR signaling differentially regulates multiple N-glycan-processing enzymes at the mRNA level to cooperatively promote β1,6GlcNAc branching, and by extension, CTLA-4 surface expression, T cell growth arrest, and self-tolerance.     

GOLPH3 keeps the Golgi residents at home

In this issue of JCB, Welch et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202106115) show that GOLPH3 mediates the sorting of numerous Golgi proteins into recycling COPI transport vesicles. This explains how many resident proteins are retained at the Golgi and reveals a key role for GOLPH3 in maintaining Golgi homeostasis.

The Golgi apparatus lies at the heart of the secretory pathway, where its major functions are the posttranslational modification of cargo proteins and lipids, particularly at the level of glycosylation, and the sorting of cargo to its correct onward destination. The Golgi is composed of stacked membrane compartments called cisternae, which contain numerous resident enzymes that act on the cargo as it passes through the organelle, from the entry or cis side to the exit or trans side. Each resident enzyme has its own distribution within the Golgi stack, resulting in the sequential modification of the secretory cargo as it moves through the Golgi.Various mechanisms exist to ensure that Golgi residents are retained within the Golgi despite the huge flux of protein and lipid through this organelle (1). Major players are COPI vesicles, which recycle Golgi residents from later to earlier cisternae, at the same time as the cisternae are thought to slowly migrate across the stack, as on a conveyor belt, progressively changing composition in a process referred to as cisternal maturation (2). Unlike the Golgi resident enzymes, which enter recycling vesicles, cargo is thought to remain within the maturing cisternae as it moves through the Golgi. Certain Golgi enzymes can bind directly to the COPI coat, explaining their inclusion in COPI vesicles (3), but for other enzymes and resident proteins, their retention mechanism is less obvious.Previous studies on the peripheral Golgi membrane protein GOLPH3 and its paralogue GOLPH3L (herein I will refer to both proteins as GOLPH3) indicated it can bind to certain Golgi enzymes and to the COPI coat, thereby acting as an adaptor to mediate sorting of these enzymes into COPI vesicles (4, 5). This was first shown for the yeast orthologue Vps74p (6, 7) and has also been demonstrated for the Drosophila version of the protein (8), consistent with a conserved function in Golgi enzyme retention. However, the extent to which GOLPH3 might participate in retention of different Golgi enzymes and other resident proteins, and its importance relative to other methods of protein retention in the Golgi, has remained unclear. Indeed, a recent study suggested that GOLPH3 selectively mediates the retention of enzymes involved in glycosphingolipid synthesis, consistent with a fairly selective role in retaining only a subset of resident Golgi enzymes (9). It should also be noted that GOLPH3 has been implicated in other functions, namely budding of exocytic vesicles from the Golgi, the DNA damage response, and mechanistic target of rapamycin signaling (10).In their current paper, Welch et al. used a combination of approaches to reassess the role of GOLPH3 at the Golgi (11). Using proteomics, they could identify numerous GOLPH3 binding partners, which included COPI, as expected, and a large number of other Golgi residents, including numerous Golgi enzymes and other membrane proteins. The ability of GOLPH3 to retain enzymes at the Golgi was confirmed using microscopy and an innovative flow cytometry–based assay to quantify surface versus Golgi abundance. The large number of possible interactors suggested that GOLPH3 could mediate the Golgi retention of many proteins. To further assess this possibility, the authors took advantage of previous observations showing that Golgi enzymes may be misrouted to the lysosome and degraded upon their failure to be retained in the Golgi (6, 7, 9). Using mass spectrometry, they could show that numerous Golgi resident proteins were depleted in GOLPH3 knockout cells, many of which were also found in the GOLPH3 interactome. This included many enzymes involved in glycosylation, consistent with GOLPH3 playing an important role in maintaining Golgi-dependent glycosylation of proteins and lipids. This was supported by lectin analysis, which showed marked changes in a broad range of glycans in the GOLPH3 knockout cells.The large number of GOLPH3 clients raises the question as to how it can recognize so many proteins. Previous work has shown binding to the cytoplasmic tails of Golgi enzymes and an interaction motif has been described for Vps74p and more recently for GOLPH3 (6, 9). However, bioinformatics analysis of the many GOLPH3 clients combined with mutational analysis, as performed in the current study, revealed the lack of a consensus sequence for GOLPH3 binding, with the common feature being a strong net positive charge combined with short cytoplasmic tail length. This would result in a high positive charge proximal to the membrane, which likely allows interaction with an acidic patch on the surface of GOLPH3. This mode of binding could mediate selective retention of many Golgi residents, while allowing for the forward trafficking of cargo proteins that have longer, less charged, or folded cytoplasmic domains.GOLPH3 is an oncogene associated with many types of cancer (12). Several mechanisms have been proposed to account for the oncogenic properties of GOLPH3, but most compelling is that changes in glycosylation are responsible. It was recently shown that GOLPH3-dependent changes in glycosphingolipids affects cell growth by altering mitogenic signaling (9). Changes in glycosylation of surface receptors has also been reported, which can affect surface abundance and hence signaling (13). The new results from Welch et al. suggest that glycosylation of many proteins and lipids may be relevant in cancer and that potentially a broad range of downstream targets contribute to oncogenesis. Such targets could influence processes beyond signaling, including cell adhesion and migration, that are known to be sensitive to changes in the surface glycome and which have been reported in previous studies on GOLPH3 (12).The study by Welch et al. indicates a major role for GOLPH3 in Golgi protein retention (Fig. 1). Clearly though, other retention mechanisms exist, including direct binding to COPI, and transmembrane domain length is also important, where the short transmembrane domain of resident proteins favors partitioning into recycling COPI vesicles and Golgi cisternal membranes of a similar thickness (1). Additional COPI adaptors are also likely, with TM9SF2 recently identified as a likely candidate, being present in Golgi vesicles and able to bind certain Golgi enzymes (1). It is possible that different resident proteins use different adaptors, or that a combination of retention mechanisms act in conjunction for certain residents, providing robustness to the retention process. However, any redundancy would seem incomplete given the strong phenotype seen upon loss of GOLPH3. GOLPH3 is localized toward the trans side of the Golgi, so it is possible that other adaptors, such as TM9SF2 and possibly others, might act earlier in the Golgi, or that direct coat binding is more important within the early Golgi. Hence different residents may be more likely to use different retention mechanisms depending on their location in the Golgi. Because GOLPH3 acts late in the Golgi and can bind many clients, we may think of it as a gatekeeper to prevent loss of numerous Golgi residents from the organelle.Open in a separate windowFigure 1.GOLPH3 plays a major role in Golgi protein retention. Golgi resident proteins, including many glycosylation enzymes, depicted by lollipops, are sorted into recycling COPI vesicles to maintain retention in the Golgi in the face of onward cisternal maturation and secretory cargo transport. Different enzymes are depicted by different lollipop shapes and colors, with GOLPH3 clients indicated by horizontal ovals. Enzymes retained by other mechanisms are depicted by lollipops with circles (transmembrane domain length), squares or vertical ovals (binding to other COPI adaptors, indicated in turquoise and purple), or hexagons (direct binding to the COPI coat). GOLPH3, which is more abundant toward the trans side of the Golgi, has many clients.With regard to possible future studies, although we have a good idea of how GOLPH3 recognizes its clients, detailed structural analysis will prove informative in elucidating how it can bind so many proteins. Similarly, identification of additional adaptors linking Golgi residents to the COPI coat will be important to generate a more comprehensive view of Golgi protein retention. Finally, in the context of disease, further analysis of the glycoproteins and glycolipids whose levels are altered because of changes in GOLPH3 expression, of which there are likely to be many, should provide significant new insights into the mechanisms underlying GOLPH3-mediated tumorigenesis.     

The role of the phosphoinositides at the Golgi complex

Eukaryotic cells are organized into a complex system of subcompartments, each with its distinct protein and lipid composition. A continuous flux of membranes crosses these compartments, and in some cases direct connections exist between the different organelles. It is thus surprising that they can maintain their individual identities. Small GTPases and the phosphoinositides have emerged as the key regulators in the maintenance of the identity of the Golgi complex. This property is due to their ability to act either alone or, more often, in combination, as cues directing and controlling the recruitment of proteins that possess phosphoinositide-binding domains. Among these many proteins there are the lipid transfer proteins, which can transfer ceramide, oxysterol, cholesterol and possibly glucosylceramide. By regulating these lipid transfer proteins in this way, this binomial combination of the small GTPases and the phosphoinositides acquires a further important role: control of the synthesis and/or distribution of other important integral constituents of cell organelles, such as the sphingolipids and cholesterol. This role is particularly relevant at the level of the Golgi complex, a key organelle in the biosynthesis, transport and sorting of both lipids and proteins that is located at the intersection of the secretory and endocytic pathways.     

The role of the phosphoinositides at the Golgi complex

The phosphorylated derivatives of phosphatidylinositol (PtdIns), known as the polyphosphoinositides (PIs), represent key membrane-localized signals in the regulation of fundamental cell processes, such as membrane traffic and cytoskeleton remodelling. The reversible production of the PIs is catalyzed through the combined activities of a number of specific phosphoinositide phosphatases and kinases that can either act separately or in concert on all the possible combinations of the 3, 4, and 5 positions of the inositol ring. So far, seven distinct PI species have been identified in mammalian cells and named according to their site(s) of phosphorylation: PtdIns 3-phosphate (PI3P); PtdIns 4-phosphate (PI4P); PtdIns 5-phosphate (PI5P); PtdIns 3,4-bisphosphate (PI3,4P2); PtdIns 4,5-bisphosphate (PI4,5P2); PtdIns 3,5-bisphosphate (PI3,5P2); and PtdIns 3,4,5-trisphosphate (PI3,4,5P3). Over the last decade, accumulating evidence has indicated that the different PIs serve not only as intermediates in the synthesis of the higher phosphorylated phosphoinositides, but also as regulators of different protein targets in their own right. These regulatory actions are mediated through the direct binding of their protein targets. In this way, the PIs can control the subcellular localization and activation of their various effectors, and thus execute a variety of cellular responses. To exert these functions, the metabolism of the PIs has to be finely regulated both in time and space, and this is achieved by controlling the subcellular distribution, regulation, and activation states of the enzymes involved in their synthesis and removal (kinases and phosphatases). These exist in many different isoforms, each of which appears to have a distinctive intracellular localization and regulation. As a consequence of this subcompartimentalized PI metabolism, a sort of "PI-fingerprint" of each cell membrane compartment is generated. When combined with the targeted recruitment of their protein effectors and the different intracellular distributions of other lipids and regulatory proteins (such as small GTPases), these factors can maintain and determine the identity of the cell organelles despite the extensive membrane flux []. Here, we provide an overview of the regulation and roles of different phosphoinositide kinases and phosphatases and their lipid products at the Golgi complex.     

Multiple activities for Arf1 at the Golgi complex

The Arf family of GTPases regulates membrane traffic and organelle structure. At the Golgi complex, Arf proteins facilitate membrane recruitment of many cytoplasmic coat proteins to allow sorting of membrane proteins for transport, stimulate the activity of enzymes that modulate the lipid composition of the Golgi, and assemble a cytoskeletal scaffold on the Golgi. Arf1 is the Arf family member most closely studied for its function at the Golgi complex. A number of regulators that activate and inactivate Arf1 on the Golgi have been described that localize to different regions of the organelle. This spatial distribution of Arf regulators may facilitate the recruitment of the coat proteins and other Arf effectors to different regions of the Golgi complex.     

Sphingomyelin synthases regulate production of diacylglycerol at the Golgi

SMS [SM (sphingomyelin) synthase] is a class of enzymes that produces SM by transferring a phosphocholine moiety on to ceramide. PC (phosphatidylcholine) is believed to be the phosphocholine donor of the reaction with consequent production of DAG (diacylglycerol), an important bioactive lipid. In the present study, by modulating SMS1 and SMS2 expression, the role of these enzymes on the elusive regulation of DAG was investigated. Because we found that modulation of SMS1 or SMS2 did not affect total levels of endogenous DAG in resting cells, whereas they produce DAG in vitro, the possibility that SMSs could modulate subcellular pools of DAG, once acute activation of the enzymes is triggered, was investigated. Stimulation of SM synthesis was induced by either treatment with short-chain ceramide analogues or by increasing endogenous ceramide at the plasma membrane, and a fluorescently labelled conventional C1 domain [from PKC (protein kinase C)] enhanced in its DAG binding activity was used to probe subcellular pools of DAG in the cell. With this approach, we found, using confocal microscopy and subcellular fractionation, that modulation of SMS1 and, to a lesser extent, SMS2 affected the formation of DAG at the Golgi apparatus. Similarly, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein PKD (protein kinase D) to the Golgi. These results provide direct evidence that both enzymes are capable of regulating the formation of DAG in cells, that this pool of DAG is biologically active, and for the first time directly implicate SMS1 and SMS2 as regulators of DAG-binding proteins in the Golgi apparatus.