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.     

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.     

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.     

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.     

Mitotic inhibition of GRASP65 organelle tethering involves Polo-like kinase 1 (PLK1) phosphorylation proximate to an internal PDZ ligand

GRASP65 links cis-Golgi cisternae via a homotypic, N-terminal PDZ interaction, and its mitotic phosphorylation disrupts this activity. Neither the identity of the PDZ ligand involved in the GRASP65 self-interaction nor the mechanism by which phosphorylation inhibits its interaction is known. Phospho-mimetic mutation of known cyclin-dependent kinase 1/cyclin B sites, all of which are in the C-terminal "regulatory domain" of the molecule, failed to block organelle tethering. However, we identified a site phosphorylated by Polo-like kinase 1 (PLK1) in the GRASP65 N-terminal domain for which mutation to aspartic acid blocked tethering and alanine substitution prevented mitotic Golgi unlinking. Further, using interaction assays, we discovered an internal PDZ ligand adjacent to the PLK phosphorylation site that was required for tethering. These results reveal the mechanism of phosphoinhibition as direct inhibition by PLK1 of the PDZ ligand underlying the GRASP65 self-interaction.     

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.     

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.     

The Yeast GRASP Grh1 Colocalizes with COPII and Is Dispensable for Organizing the Secretory Pathway

In mammalian cells, the ‘Golgi reassembly and stacking protein’ (GRASP) family has been implicated in Golgi stacking, but the broader functions of GRASP proteins are still unclear. The yeast Saccharomyces cerevisiae contains a single non‐essential GRASP homolog called Grh1. However, Golgi cisternae in S. cerevisiae are not organized into stacks, so a possible structural role for Grh1 has been difficult to test. Here, we examined the localization and function of Grh1 in S. cerevisiae and in the related yeast Pichia pastoris, which has stacked Golgi cisternae. In agreement with earlier studies indicating that Grh1 interacts with coat protein II (COPII) vesicle coat proteins, we find that Grh1 colocalizes with COPII at transitional endoplasmic reticulum (tER) sites in both yeasts. Deletion of P. pastoris Grh1 had no obvious effect on the structure of tER–Golgi units. To test the role of S. cerevisiae Grh1, we exploited the observation that inhibiting ER export in S. cerevisiae generates enlarged tER sites that are often associated with the cis Golgi. This tER–Golgi association was preserved in the absence of Grh1. The combined data suggest that Grh1 acts early in the secretory pathway, but is dispensable for the organization of secretory compartments.     

Mapping the interaction between GRASP65 and GM130, components of a protein complex involved in the stacking of Golgi cisternae.

The nature of the complex containing GRASP65, a membrane protein involved in establishing the stacked structure of the Golgi apparatus, and GM130, a putative Golgi matrix protein and vesicle docking receptor, was investigated. Gel filtration revealed that GRASP65 and GM130 interact in detergent extracts of Golgi membranes under both interphase and mitotic conditions, and that this complex can bind to the vesicle docking protein p115. Using in vitro translation and site-directed mutagenesis in conjunction with immunoprecipitation, the binding site for GRASP65 on GM130 was mapped to the sequence xxNDxxxIMVI-COOH at the C-terminus of GM130, a region known to be required for its localization to the Golgi apparatus. The same approach was used to show that the binding site for GM130 on GRASP65 maps to amino acids 189-201, a region conserved in the mammalian and yeast proteins and reminiscent of PDZ domains. Using green fluorescent protein (GFP)-tagged reporter constructs, it was shown that one essential function of the interaction between GRASP65 and GM130 is in the correct targeting of the two proteins to the Golgi apparatus.     

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.     

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.     

SNARE status regulates tether recruitment and function in homotypic COPII vesicle fusion

In mammals, coat complex II (COPII)-coated transport vesicles deliver secretory cargo to vesicular tubular clusters (VTCs) that facilitate cargo sorting and transport to the Golgi. We documented in vitro tethering and SNARE-dependent homotypic fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers characteristic of VTCs ( Xu, D., and Hay, J. C. (2004) J. Cell Biol. 167, 997-1003). COPII vesicles thus appear to contain all necessary components for homotypic tethering and fusion, providing a pathway for de novo VTC biogenesis. Here we demonstrate that antibodies against the endoplasmic reticulum/Golgi SNARE Syntaxin 5 inhibit COPII vesicle homotypic tethering as well as fusion, implying an unanticipated role for SNAREs upstream of fusion. Inhibition of SNARE complex access and/or disassembly with dominant-negative alpha-soluble NSF attachment protein (SNAP) also inhibited tethering, implicating SNARE status as a critical determinant in COPII vesicle tethering. The tethering-defective vesicles generated in the presence of dominant-negative alpha-SNAP specifically lacked the Rab1 effectors p115 and GM130 but not other peripheral membrane proteins. Furthermore, Rab effectors, including p115, were shown to be required for homotypic COPII vesicle tethering. Thus, our results demonstrate a requirement for SNARE-dependent tether recruitment and function in COPII vesicle fusion. We anticipate that recruitment of tether molecules by an upstream SNARE signal ensures that tethering events are initiated only at focal sites containing appropriately poised fusion machinery.     

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.     

Characterization of a cis-Golgi matrix protein, GM130

Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER- Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.     

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.     

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.     

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.     

Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p

Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly. However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown. We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi. This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst. Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p. All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN. vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role. Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p. The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery.     

Structure of the membrane-tethering GRASP domain reveals a unique PDZ ligand interaction that mediates Golgi biogenesis

Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.     

Coordination of COPII vesicle trafficking by Sec23

Coat protein complex II (COPII) is a multi-subunit protein complex responsible for the formation of membrane vesicles at the endoplasmic reticulum. The assembly of this complex on the endoplasmic reticulum membrane needs to be tightly regulated to ensure efficient and specific incorporation of cargo proteins into nascent vesicles. Recent studies of a genetic disease affecting COPII function, and a structural analysis of COPII subunit interactions emphasize the central role of the Sec23 subunit in COPII coat assembly. Similarly, the demonstration that Sec23 interacts physically and functionally with proteins involved in both vesicle tethering and the transport along microtubules indicates that the Sec23 subunit is crucially important in linking COPII vesicle formation to anterograde transport events.