GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system.

We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery. GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae. GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi. Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65. These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae.     

A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic.

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.     

Structural Basis for the Interaction between the Golgi Reassembly-stacking Protein GRASP65 and the Golgi Matrix Protein GM130

GM130 and GRASP65 are Golgi peripheral membrane proteins that play a key role in Golgi stacking and vesicle tethering. However, the molecular details of their interaction and their structural role as a functional unit remain unclear. Here, we present the crystal structure of the PDZ domains of GRASP65 in complex with the GM130 C-terminal peptide at 1.96-Å resolution. In contrast to previous findings proposing that GM130 interacts with GRASP65 at the PDZ2 domain only, our crystal structure of the complex indicates that GM130 binds to GRASP65 at two distinct sites concurrently and that both the PDZ1 and PDZ2 domains of GRASP65 participate in this molecular interaction. Mutagenesis experiments support these structural observations and demonstrate that they are required for GRASP65-GM130 association.     

Dynamics of Golgi matrix proteins after the blockage of ER to Golgi transport

When the ER to Golgi transport is blocked by a GTP-restricted mutant of Sar1p (H79G) in NRK-52E cells, most Golgi resident proteins are transported back into the ER. In contrast, the cis-Golgi matrix proteins GM130 and GRASP65 are retained in punctate cytoplasmic structures, namely Golgi remnants. Significant amounts of the medial-Golgi matrix proteins golgin-45, GRASP55 and giantin are retained in the Golgi remnants, but a fraction of these proteins relocates to the ER. Golgin-97, a candidate trans-Golgi network matrix protein, is retained in Golgi remnant-like structures, but mostly separated from GM130 and GRASP65. Interestingly, most Sec13p, a COPII component, congregates into larger cytoplasmic clusters soon after the microinjection of Sar1p(H79G), and these move to accumulate around the Golgi apparatus. Sec13p clusters remain associated with Golgi remnants after prolonged incubation. Electron microscopic analysis revealed that Golgi remnants are clusters of larger vesicles with smaller vesicles, many of which are coated. GM130 is mainly associated with larger vesicles and Sec13p with smaller coated vesicles. The Sec13p clusters disperse when p115 binding to the Golgi apparatus is inhibited. These results suggest that cis-Golgi matrix proteins resist retrograde transport flow and stay as true residents in Golgi remnants after the inhibition of ER to Golgi transport.     

Organelle tethering by a homotypic PDZ interaction underlies formation of the Golgi membrane network

Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.     

A Role for Giantin in Docking COPI Vesicles to Golgi Membranes

We have previously shown that p115, a vesicle docking protein, binds to two proteins (p130 and p400) in detergent extracts of Golgi membranes. p130 was identified as GM130, a Golgi matrix protein, and was shown to act as a membrane receptor for p115. p400 has now been identified as giantin, a Golgi membrane protein with most of its mass projecting into the cytoplasm. Giantin is found on COPI vesicles and pretreatment with antibodies inhibits both the binding of p115 and the docking of these vesicles with Golgi membranes. In contrast, GM130 is depleted from COPI vesicles and inhibition of the GM130 on Golgi membranes, using either antibodies or an NH₂-terminal GM130 peptide, inhibits p115 binding and vesicle docking. Together these results suggest that COPI vesicles are docked by giantin on the COPI vesicles and GM130 on Golgi membranes with p115 providing a bridge.     

Rapid degradation of GRASP55 and GRASP65 reveals their immediate impact on the Golgi structure

GRASP55 and GRASP65 have been implicated in stacking of Golgi cisternae and lateral linking of stacks within the Golgi ribbon. However, RNAi or gene knockout approaches to dissect their respective roles have often resulted in conflicting conclusions. Here, we gene-edited GRASP55 and/or GRASP65 with a degron tag in human fibroblasts, allowing for induced rapid degradation by the proteasome. We show that acute depletion of either GRASP55 or GRASP65 does not affect the Golgi ribbon, while chronic degradation of GRASP55 disrupts lateral connectivity of the ribbon. Acute double depletion of both GRASPs coincides with the loss of the vesicle tethering proteins GM130, p115, and Golgin-45 from the Golgi and compromises ribbon linking. Furthermore, GRASP55 and/or GRASP65 is not required for maintaining stacks or de novo assembly of stacked cisternae at the end of mitosis. These results demonstrate that both GRASPs are dispensable for Golgi stacking but are involved in maintaining the integrity of the Golgi ribbon together with GM130 and Golgin-45.     

GM130 and GRASP65-dependent lateral cisternal fusion allows uniform Golgi-enzyme distribution

The mammalian Golgi apparatus exists as stacks of cisternae that are laterally linked to form a continuous membrane ribbon, but neither the molecular requirements for, nor the purpose of, Golgi ribbon formation are known. Here, we demonstrate that ribbon formation is mediated by specific membrane-fusion events that occur during Golgi assembly, and require the Golgi proteins GM130 and GRASP65. Furthermore, these GM130 and GRASP65-dependent lateral cisternal-fusion reactions are necessary to achieve uniform distribution of enzymes in the Golgi ribbon. The membrane continuity created by ribbon formation facilitates optimal processing conditions in the biosynthetic pathway.     

Golgi fragmentation during Fas-mediated apoptosis is associated with the rapid loss of GM130

During apoptosis, the Golgi complex becomes fragmented and key proteins (e.g., GRASP65 and p115) are targets for caspase cleavage. GM130, an integral membrane protein, contributes to the maintenance of Golgi structure and facilitates membrane fusion with secretory vesicles. We show that GM130 levels decrease during Fas-induced apoptosis but not during staurosporine-induced apoptosis while in both models p115 levels remain unaffected. We conclude that GM130 is rapidly diminished during Fas-mediated apoptosis associated with Golgi fragmentation in contrast to previous studies which have suggested that loss of GM130 during apoptosis is a late event.     

The yeast orthologue of GRASP65 forms a complex with a coiled-coil protein that contributes to ER to Golgi traffic

The mammalian Golgi protein GRASP65 is required in assays that reconstitute cisternal stacking and vesicle tethering. Attached to membranes by an N-terminal myristoyl group, it recruits the coiled-coil protein GM130. The relevance of this system to budding yeasts has been unclear, as they lack an obvious orthologue of GM130, and their only GRASP65 relative (Grh1) lacks a myristoylation site and has even been suggested to act in a mitotic checkpoint. In this study, we show that Grh1 has an N-terminal amphipathic helix that is N-terminally acetylated and mediates association with the cis-Golgi. We find that Grh1 forms a complex with a previously uncharacterized coiled-coil protein, Ydl099w (Bug1). In addition, Grh1 interacts with the Sec23/24 component of the COPII coat. Neither Grh1 nor Bug1 are essential for growth, but biochemical assays and genetic interactions with known mediators of vesicle tethering (Uso1 and Ypt1) suggest that the Grh1-Bug1 complex contributes to a redundant network of interactions that mediates consumption of COPII vesicles and formation of the cis-Golgi.     

A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi cisternae in a cell-free system.

During telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.     

Phosphorylation of the vesicle-tethering protein p115 by a casein kinase II-like enzyme is required for Golgi reassembly from isolated mitotic fragments

Coat protein I (COPI) transport vesicles can be tethered to Golgi membranes by a complex of fibrous, coiled-coil proteins comprising p115, Giantin and GM130. p115 has been postulated to act as a bridge, linking Giantin on the vesicle to GM130 on the Golgi membrane. Here we show that the acidic COOH terminus of p115 mediates binding to both GM130 and Giantin as well as linking the two together. Phosphorylation of serine 941 within this acidic domain enhances the binding as well as the link between them. Phosphorylation is mediated by casein kinase II (CKII) or a CKII-like kinase. Surprisingly, the highly conserved NH(2)-terminal head domain of p115 is not required for the NSF (N-ethylmaleimide-sensitive fusion protein)-catalyzed reassembly of cisternae from mitotic Golgi fragments in a cell-free system. However, the ability of p115 to link GM130 to Giantin and the phosphorylation of p115 at serine 941 are required for NSF-catalyzed cisternal regrowth. p115 phosphorylation may be required for the transition from COPI vesicle tethering to COPI vesicle docking, an event that involves the formation of trans-SNARE [corrected] (trans-soluble NSF attachment protein [SNAP] receptor) complexes.     

Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner

During membrane traffic, transport carriers are first tethered to the target membrane prior to undergoing fusion. Mechanisms exist to connect tethering with fusion, but in most cases, the details remain poorly understood. GM130 is a member of the golgin family of coiled-coil proteins tat is involved in membrane tethering at the endoplasmic reticulum (ER) to Golgi intermediate compartment and cis-Golgi. Here, we demonstrate that GM130 interacts with syntaxin 5, a t-SNARE also localized to the early secretory pathway. Binding to syntaxin 5 is specific, direct, and mediated by the membrane-proximal region of GM130. Interestingly, interaction with syntaxin 5 is inhibited by the binding of the vesicle docking protein p115 to a distal binding site in GM130. The interaction between GM130 and the small GTPase Rab1 is also inhibited by p115 binding. Our findings suggest a mechanism for coupling membrane tethering and fusion at the ER to Golgi intermediate compartment and cis-Golgi, with GM130 playing a central role in linking these processes. Consistent with this hypothesis, we find that depletion of GM130 by RNA interference slows the rate of ER to Golgi trafficking in vivo. The interactions of GM130 with syntaxin 5 and Rab1 are also regulated by mitotic phosphorylation, which is likely to contribute to the inhibition of ER to Golgi trafficking that occurs when mammalian cells enter mitosis.     

EspG of enteropathogenic and enterohemorrhagic E. coli binds the Golgi matrix protein GM130 and disrupts the Golgi structure and function

The enteric pathogens enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Shigella flexneri all translocate at least one effector protein of the EspG protein family into host cells via a type III secretion system (T3SS). The EspG family comprises EspG, EspG2 and VirA. From a Y2H screen, we identified the Golgi matrix protein GM130 as a potential binding partner of EspG. We confirmed EspG:GM130 protein interaction by affinity co-purification. In co-immunoprecipitation experiments EspG was co-precipitated with GM130 while both GM130 and tubulins were co-precipitated with EspG. When expressed ectopically in HeLa cells, the EspG protein family all localized to the Golgi and induced fragmentation of the Golgi apparatus. All EspG family proteins were also able to disrupt protein secretion to a greater extent than the T3SS effector NleA/EspI, which has previously been shown to localize to the Golgi and interact with SEC24 to disrupt COPII vesicle formation. We hypothesize that EspG:GM130 interaction disrupts protein secretion either through direct disruption of GM130 function or through recruitment of other EspG interacting proteins to the Golgi.     

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.     

Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis -SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444–448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis -Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis -Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.     

Golgi dynamics during meiosis are distinct from mitosis and are coupled to endoplasmic reticulum dynamics until fertilization

One current theory of the Golgi apparatus views its organization as containing both a matrix fraction of structural proteins and a reservoir of cycling enzymes. During mitosis, the putative matrix protein GM130 is phosphorylated and relocalized to spindle poles. When the secretory pathway is inhibited during interphase, GM130 redistributes to regions adjacent to vesicle export sites on the endoplasmic reticulum (ER). Strikingly, meiotic maturation and fertilization in nonrodent mammalian eggs presents a unique experimental environment for the Golgi apparatus, because secretion is inhibited until after fertilization, and because the centrosome is absent until introduced by the sperm. Here, we test the hypothesis that phosphorylated GM130 associates not with meiotic spindle poles, but with ER clusters in the mature bovine oocyte. At the germinal vesicle stage, phosphorylated GM130 is observed as fragments dispersed throughout the cytoplasm. During meiotic maturation, GM130 reorganizes into punctate foci that associate near the ER-resident protein calreticulin and is notably absent from the meiotic spindle. GM130 colocalizes with Sec23, a marker for ER vesicle export sites, but not with Lens culinaris agglutinin, a marker for cortical granules. Because disruption of vesicle transport has been shown to block meiotic maturation and embryonic cleavage in some species, we also test the hypothesis that fertilization and cytokinesis are inhibited with membrane trafficking disruptor brefeldin A (BFA). Despite Golgi fragmentation after BFA treatment, pronuclei form and unite, and embryos cleave and develop through the eight-cell stage. We conclude that, while the meiotic phosphorylation cycle of GM130 mirrors that of mitosis, absence of a maternal centrosome precludes Golgi association with the meiotic spindle. Fertilization introduces the sperm centrosome that can reorganize Golgi proteins, but neither fertilization nor cytokinesis prior to compaction requires a functional Golgi apparatus.     

Binding relationships of membrane tethering components. The giantin N terminus and the GM130 N terminus compete for binding to the p115 C terminus

By forming a molecular tether between two membranes, p115, giantin, and GM130 may mediate multiple Golgi-related processes including vesicle transport, cisternae formation, and cisternal stacking. The tether is proposed to involve the simultaneous binding of p115 to giantin on one membrane and to GM130 on another membrane. To explore this model, we tested for the presence of the putative giantin-p115-GM130 ternary complex. We first mapped p115-binding site in giantin to a 70-amino acid coiled-coil domain at the extreme N terminus, a position that may exist up to 400 nm away from the Golgi membrane. We then generated glutathione S-transferase (GST) fusion proteins containing either giantin's or GM130's p115 binding site and tested whether such proteins could bind p115 and GM130 or bind p115 and giantin, respectively. Unexpectedly, GST fusions containing either the giantin or the GM130 p115 binding site efficiently bound p115, but the p115 bound to GST-giantin did not bind GM130, and the p115 bound to GST-GM130 did not bind giantin. To explain this result, we mapped the giantin binding site in p115 and found that it is located at the C-terminal acidic domain, the same domain involved in binding GM130. The presence of a single binding site in p115 for giantin and GM130 was confirmed by demonstration that giantin and GM130 compete for binding to p115. These results question a simple tethering model involving a ternary giantin-p115-GM130 complex and suggest that p115-giantin and p115-GM130 interactions might mediate independent membrane tethering events.     

Dynamic nucleation of Golgi apparatus assembly from the endoplasmic reticulum in interphase hela cells

Models of Golgi apparatus biogenesis and maintenance are focused on two possibilities: one is self-assembly from the endoplasmic reticulum, and the other is nucleation by a stable template. Here, we asked in three different experimental situations whether assembly of the Golgi apparatus might be dynamically nucleated. During microtubule depolymerization, the integral membrane protein p27 and the peripheral Golgi protein GM130, appeared in newly formed, scattered Golgi elements before three different Golgi apparatus cisternal enzymes, whereas GRASP55, a medial peripheral Golgi protein, showed, if anything, a tendency to accumulate in scattered Golgi elements later than a cisternal enzyme. During Golgi formation after brefeldin A washout, endoplasmic reticulum exit of Golgi resident enzymes could be completely separated from that of p27 and GM130. p27 and GM130 accumulation was onto newly organized perinuclear structures, not brefeldin A remnants, and preceded that of a cisternal enzyme. Reassembly was completely sensitive to guanosine 5'-diphosphate-restricted Sar1p. When cells were microinjected with Sar1pWT DNA to reverse a guanosine 5'-diphosphate-restricted Sar1p endoplasmic reticulum-exit block phenotype, GM130 and p27 collected perinuclearly with little to no exit of a cisternal enzyme from the endoplasmic reticulum. The overall data strongly indicate that the assembly of the Golgi apparatus can be nucleated dynamically by GM130/p27 associated structures. We define dynamic nucleation as the first step in a staged organelle assembly process in which new component association forms a microscopically visible structure onto which other components add later, e.g. Golgi cisternae.     

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.