Golgins and GRASPs: Holding the Golgi together

The GRASP and golgin families of proteins have emerged as key components of the Golgi apparatus, with major roles in both the structural organisation of this organelle and the trafficking that occurs there. Both types of protein participate in membrane tethering events that occur upstream of membrane fusion as well as contributing to the structural scaffold that defines Golgi architecture, referred to as the Golgi matrix. The importance of these proteins is highlighted by their targeting in mitosis, apoptosis, and pathogenic infections that cause dramatic structural and functional reorganisation of the Golgi apparatus. In this review we will discuss our current understanding of GRASP and golgin function, highlighting some of the common themes that have emerged as well as describing previously unsuspected roles for these proteins in various cellular processes.     

YSK1 is activated by the Golgi matrix protein GM130 and plays a role in cell migration through its substrate 14-3-3zeta

The Golgi apparatus has long been suggested to be important for directing secretion to specific sites on the plasma membrane in response to extracellular signaling events. However, the mechanisms by which signaling events are coordinated with Golgi apparatus function remain poorly understood. Here, we identify a scaffolding function for the Golgi matrix protein GM130 that sheds light on how such signaling events may be regulated. We show that the mammalian Ste20 kinases YSK1 and MST4 target to the Golgi apparatus via the Golgi matrix protein GM130. In addition, GM130 binding activates these kinases by promoting autophosphorylation of a conserved threonine within the T-loop. Interference with YSK1 function perturbs perinuclear Golgi organization, cell migration, and invasion into type I collagen. A biochemical screen identifies 14-3-3zeta as a specific substrate for YSK1 that localizes to the Golgi apparatus, and potentially links YSK1 signaling at the Golgi apparatus with protein transport events, cell adhesion, and polarity complexes important for cell migration.     

Regulation of Cell Death by Recycling Endosomes and Golgi Membrane Dynamics via a Pathway Involving Src-family kinases,Cdc42 and Rab11a

Actin dynamics and membrane trafficking influence cell commitment to programmed cell death through largely undefined mechanisms. To investigate how actin and recycling endosome (RE) trafficking can engage death signaling, we studied the death program induced by the adenovirus early region 4 open reading frame 4 (E4orf4) protein as a model. We found that in the early stages of E4orf4 expression, Src-family kinases (SFKs), Cdc42, and actin perturbed the organization of the endocytic recycling compartment and promoted the transport of REs to the Golgi apparatus, while inhibiting recycling of protein cargos to the plasma membrane. The resulting changes in Golgi membrane dynamics that relied on actin-regulated Rab11a membrane trafficking triggered scattering of Golgi membranes and contributed to the progression of cell death. A similar mobilization of RE traffic mediated by SFKs, Cdc42 and Rab11a also contributed to Golgi fragmentation and to cell death progression in response to staurosporine, in a caspase-independent manner. Collectively, these novel findings suggest that diversion of RE trafficking to the Golgi complex through a pathway involving SFKs, Cdc42, and Rab11a plays a general role in death signaling by mediating regulated changes in Golgi dynamics.     

The Golgi complex: a hub of the secretory pathway

The Golgi complex plays a central role in protein secretion by regulating cargo sorting and trafficking. As these processes are of functional importance to cell polarity, motility, growth, and division, there is considerable interest in achieving a comprehensive understanding of Golgi complex biology. However, the unique stack structure of this organelle has been a major hurdle to our understanding of how proteins are secreted through the Golgi apparatus. Herein, we summarize available relevant research to gain an understanding of protein secretion via the Golgi complex. This includes the molecular mechanisms of intra-Golgi trafficking and cargo export in the trans-Golgi network. Moreover, we review recent insights on signaling pathways regulated by the Golgi complex and their physiological significance.     

Golgins and GTPases, giving identity and structure to the Golgi apparatus

In this review we will focus on the recent advances in how coiled-coil proteins of the golgin family give identity and structure to the Golgi apparatus in animal cells. A number of recent studies reveal a common theme for the targeting of golgins containing the ARL-binding GRIP domain, and the related ARF-binding GRAB domain. Similarly, other golgins such as the vesicle tethering factor p115 and Bicaudal-D are targeted by the Rab GTPases, Rab1 and Rab6, respectively. Together golgins and their regulatory GTPases form a complex network, commonly known as the Golgi matrix, which organizes Golgi membranes and regulates membrane trafficking.     

Maintenance of Golgi structure and function depends on the integrity of ER export.

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.     

Golgins in the structure and dynamics of the Golgi apparatus

Golgins are a family of coiled-coil proteins associated with the Golgi apparatus necessary for tethering events in membrane fusion and as structural supports for Golgi cisternae. Recent work has shown that golgins such as GM130, golgin-45 and p115 bind to Rab GTPases via their coiled-coil domains, and that GM130, rather than being part of a static structural matrix, is in dynamic exchange between the membrane surface and the cytoplasm. Golgins such as bicaudal-D1 and -D2 bind to Rab6, but, rather than tethering membranes together, link vesicles to the cytoskeleton, thus adding a new function for this class of proteins. Other golgins containing the Golgi targeting GRIP domain, rather than binding Rabs, interact with and are recruited to membranes by another class of GTPase, the Arls. Current evidence therefore suggests that golgins function in a variety of membrane-membrane and membrane-cytoskeleton tethering events at the Golgi apparatus, and that all these are regulated by small GTPases of the Rab and Arl families.     

Golgi Positioning

The Golgi apparatus in mammalian cells is positioned near the centrosome-based microtubule-organizing center (Fig. 1). Secretory cargo moves inward in membrane carriers for delivery to Golgi membranes in which it is processed and packaged for transport outward to the plasma membrane. Cytoplasmic dynein motor proteins (herein termed dynein) primarily mediate inward cargo carrier movement and Golgi positioning. These motors move along microtubules toward microtubule minus-ends embedded in centrosomes. Centripetal motility is controlled by a host of regulators whose precise functions remain to be determined. Significantly, a specific Golgi receptor for dynein has not been identified. This has impaired progress toward elucidation of membrane-motor-microtubule attachment in the periphery and, after inward movement, recycling of the motor for another round. Pericentrosomal positioning of the Golgi apparatus is dynamic. It is regulated during critical cellular processes such as mitosis, differentiation, cell polarization, and cell migration. Positioning is also important as it aligns the Golgi along an axis of cell polarity. In certain cell types, this promotes secretion directed to the proximal plasma membrane domain thereby maintaining specializations critical for diverse processes including wound healing, immunological synapse formation, and axon determination.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

Membrane protein transport between the endoplasmic reticulum and the Golgi in tobacco leaves is energy dependent but cytoskeleton independent: evidence from selective photobleaching

The mechanisms that control protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus are poorly characterized in plants. Here, we examine in tobacco leaves the structural relationship between Golgi and ER membranes using electron microscopy and demonstrate that Golgi membranes contain elements that are in close association and/or in direct contact with the ER. We further visualized protein trafficking between the ER and the Golgi using Golgi marker proteins tagged with green fluorescent protein. Using photobleaching techniques, we showed that Golgi membrane markers constitutively cycle to and from the Golgi in an energy-dependent and N-ethylmaleimide-sensitive manner. We found that membrane protein transport toward the Golgi occurs independently of the cytoskeleton and does not require the Golgi to be motile along the surface of the ER. Brefeldin A treatment blocked forward trafficking of Golgi proteins before their redistribution into the ER. Our results indicate that in plant cells, the Golgi apparatus is a dynamic membrane system whose components continuously traffic via membrane trafficking pathways regulated by brefeldin A- and N-ethylmaleimide-sensitive machinery.     

Structural organization of the Golgi apparatus

The Golgi apparatus is a universal feature of eukaryotes, carrying out the key functions of processing, sorting and trafficking of newly synthesized membrane and secretory proteins. The Golgi apparatus has a clearly defined structure, comprising stacks of flattened cisternal membranes that in vertebrates are connected to form a ribbon. How this structure is maintained and how it relates to the functions of the Golgi apparatus has long been an area of interest. In this review I describe recent progress in the identification and characterization of the molecular machinery that together help generate the characteristic organization of this organelle.     

Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus.

The Golgi apparatus is a highly complex organelle comprised of a stack of cisternal membranes on the secretory pathway from the ER to the cell surface. This structure is maintained by an exoskeleton or Golgi matrix constructed from a family of coiled-coil proteins, the golgins, and other peripheral membrane components such as GRASP55 and GRASP65. Here we find that TMP21, p24a, and gp25L, members of the p24 cargo receptor family, are present in complexes with GRASP55 and GRASP65 in vivo. GRASPs interact directly with the cytoplasmic domains of specific p24 cargo receptors depending on their oligomeric state, and mutation of the GRASP binding site in the cytoplasmic tail of one of these, p24a, results in it being transported to the cell surface. These results suggest that one function of the Golgi matrix is to aid efficient retention or sequestration of p24 cargo receptors and other membrane proteins in the Golgi apparatus.     

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.     

Decoupling Polarization of the Golgi Apparatus and GM1 in the Plasma Membrane

Cell polarization is a process of coordinated cellular rearrangements that prepare the cell for migration. GM1 is synthesized in the Golgi apparatus and localized in membrane microdomains that appear at the leading edge of polarized cells, but the mechanism by which GM1 accumulates asymmetrically is unknown. The Golgi apparatus itself becomes oriented toward the leading edge during cell polarization, which is thought to contribute to plasma membrane asymmetry. Using quantitative image analysis techniques, we measure the extent of polarization of the Golgi apparatus and GM1 in the plasma membrane simultaneously in individual cells subject to a wound assay. We find that GM1 polarization starts just 10 min after stimulation with growth factors, while Golgi apparatus polarization takes 30 min. Drugs that block Golgi polarization or function have no effect on GM1 polarization, and, conversely, inhibiting GM1 polarization does not affect Golgi apparatus polarization. Evaluation of Golgi apparatus and GM1 polarization in single cells reveals no correlation between the two events. Our results indicate that Golgi apparatus and GM1 polarization are controlled by distinct intracellular cascades involving the Ras/Raf/MEK/ERK and the PI3K/Akt/mTOR pathways, respectively. Analysis of cell migration and invasion suggest that MEK/ERK activation is crucial for two dimensional migration, while PI3K activation drives three dimensional invasion, and no cumulative effect is observed from blocking both simultaneously. The independent biochemical control of GM1 polarity by PI3K and Golgi apparatus polarity by MEK/ERK may act synergistically to regulate and reinforce directional selection in cell migration.     

Sequential Depletion and Acquisition of Proteins during Golgi Stack Disassembly and Reformation

Herein, we report the stepwise transport of multiple plant Golgi membrane markers during disassembly of the Golgi apparatus in tobacco leaf epidermal cells in response to the induced expression of the GTP‐locked Sar1p or Brefeldin A (BFA), and reassembly on BFA washout. The distribution of fluorescent Golgi‐resident N‐glycan processing enzymes and matrix proteins (golgins) with specific cis–trans‐Golgi sub‐locations was followed by confocal microscopy during disassembly and reassembly. The first event during Golgi disassembly was the loss of trans‐Golgi enzymes and golgins from Golgi membranes, followed by a sequential redistribution of medial and cis‐Golgi enzymes into the endoplasmic reticulum (ER), whilst golgins were relocated to the ER or cytoplasm. This event was confirmed by fractionation and immuno‐blotting. The sequential redistribution of Golgi components in a trans–cis sequence may highlight a novel retrograde trafficking pathway between the trans‐Golgi and the ER in plants. Release of Golgi markers from the ER upon BFA washout occurred in the opposite sequence, with cis‐matrix proteins labelling Golgi‐like structures before cis/medial enzymes. Trans‐enzyme location was preceded by trans‐matrix proteins being recruited back to Golgi membranes. Our results show that Golgi disassembly and reassembly occur in a highly ordered fashion in plants.     

Characterization of Golgi scaffold proteins and their roles in compartmentalizing cell signaling

Subcellular compartmentalization has become an important theme in cell signaling. In particular, the Golgi apparatus (GA) plays a prominent role in compartmentalizing signaling cascades that originate at the plasma membrane or other organelles. To precisely regulate this process, cells have evolved a unique class of organizer proteins, termed “scaffold proteins”. Sef, PAQR3, PAQR10 and PAQR11 are scaffold proteins that have recently been identified on the GA and are referred to as Golgi scaffolds. The major cell growth signaling pathways, such as Ras/MAPK, PI3K/AKT, insulin and VEGF (vascular endothelial growth factor), are tightly regulated spatially and temporally by these Golgi scaffolds to ensure a physiologically appropriate outcome. Here, we discuss the subcellular localization and characterization of the topology and functional domains of these Golgi scaffolds and summarize their roles in the compartmentalization of cell signaling. We also highlight the physiological and pathological roles of these Golgi scaffolds in tumorigenesis and developmental disorders.     

Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond

Significant advances have been made in recent years that have increased our understanding of the trafficking to and from membranes that are functionally linked to the Golgi apparatus in plants. New routes from the Golgi to organelles outside the secretory pathway are now being identified, revealing the importance of the Golgi apparatus as a major sorting station in the plant cell. This review discusses our current perception of Golgi structure and organization as well as the molecular mechanisms that direct traffic in and out of the Golgi.     

Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension

Monocrotaline (MCT)-induced pulmonary hypertension (PH) in the rat is a widely used experimental model. We have previously shown that MCT pyrrole (MCTP) produces loss of caveolin-1 (cav-1) and endothelial nitric oxide synthase from plasma membrane raft microdomains in pulmonary arterial endothelial cells (PAEC) with the trapping of these proteins in the Golgi organelle (the Golgi blockade hypothesis). In the present study, we investigated the mechanisms underlying this intracellular trafficking block in experiments in cell culture and in the MCT-treated rat. In cell culture, PAEC showed trapping of cav-1 in Golgi membranes as early as 6 h after exposure to MCTP. Phenotypic megalocytosis and a reduction in anterograde trafficking (assayed in terms of the secretion of horseradish peroxidase derived from exogenously transfected expression constructs) were evident within 12 h after MCTP. Cell fractionation and immunofluorescence techniques revealed the marked accumulation of diverse Golgi tethers, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), and soluble NSF attachment proteins (SNAPs), which mediate membrane fusion during vesicular trafficking (GM130, p115, giantin, golgin 84, clathrin heavy chain, syntaxin-4, -6, Vti1a, Vti1b, GS15, GS27, GS28, SNAP23, and alpha-SNAP) in the enlarged/circumnuclear Golgi in MCTP-treated PAEC and A549 lung epithelial cells. Moreover, NSF, an ATPase required for the "disassembly" of SNARE complexes subsequent to membrane fusion, was increasingly sequestered in non-Golgi membranes. Immunofluorescence studies of lung tissue from MCT-treated rats confirmed enlargement of perinuclear Golgi elements in lung arterial endothelial and parenchymal cells as early as 4 days after MCT. Thus MCT-induced PH represents a disease state characterized by dysfunction of Golgi tethers, SNAREs, and SNAPs and of intracellular vesicular trafficking.     

Association of a novel PDZ domain-containing peripheral Golgi protein with the Q-SNARE (Q-soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) protein syntaxin 6

PDZ domains are involved in the scaffolding and assembly of multi-protein complexes at various subcellular sites. We describe here the isolation and characterization of a novel PDZ domain-containing protein that localizes to the Golgi apparatus. Using an in silico cloning approach, we have identified and isolated a cDNA encoding a ubiquitously expressed 59-kDa protein that we call FIG. It is composed of two coiled coil regions, a leucine zipper, and a single PDZ domain. Cytological studies using indirect immunofluorescence microscopy revealed that FIG is a peripheral protein that uses one of its coiled coil domains to localize to the Golgi apparatus. To ascertain the modalities of this Golgi localization, the same coiled coil region was tested for its ability to interact with a panel of coiled coil domain-containing integral membrane Golgi proteins. Using a series of GST fusion protein binding assays, co-immunofluorescence and co-immunoprecipitation experiments, we show that FIG specifically binds to the coiled coil domain-containing Q-SNARE (Q-soluble NSF attachment protein receptor) protein syntaxin 6 both in vitro and in vivo. The structural features of FIG and its interaction with a SNARE protein suggest that FIG may play a role in membrane vesicle trafficking. This is the first example of a PDZ domain-containing peripheral protein that localizes to the Golgi through a coiled coil-mediated interaction with a resident membrane protein. Our results broaden the scope of PDZ domain-mediated functions.