A Highlights from MBoC Selection: Coupling of vesicle tethering and Rab binding is required for in vivo functionality of the golgin GMAP-210

Golgins are extended coiled-coil proteins believed to participate in membrane-tethering events at the Golgi apparatus. However, the importance of golgin-mediated tethering remains poorly defined, and alternative functions for golgins have been proposed. Moreover, although golgins bind to Rab GTPases, the functional significance of Rab binding has yet to be determined. In this study, we show that depletion of the golgin GMAP-210 causes a loss of Golgi cisternae and accumulation of numerous vesicles. GMAP-210 function in vivo is dependent upon its ability to tether membranes, which is mediated exclusively by the amino-terminal ALPS motif. Binding to Rab2 is also important for GMAP-210 function, although it is dispensable for tethering per se. GMAP-210 length is also functionally important in vivo. Together our results indicate a key role for GMAP-210–mediated membrane tethering in maintaining Golgi structure and support a role for Rab2 binding in linking tethering with downstream docking and fusion events at the Golgi apparatus.     

Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins

Vesicles and other carriers destined for the Golgi apparatus must be guided to the correct cisternae. Golgins, long coiled-coil proteins that localize to particular Golgi subdomains via their C termini, are candidate regulators of vesicle sorting. In this study, we report that the GRIP domain golgins, whose C termini bind the Arf-like 1 G protein on the trans-Golgi, can also bind four members of the Rab family of G proteins. The Rab2-, Rab6-, Rab19-, and Rab30-binding sites are within the coiled-coil regions that are not required for Golgi targeting. Binding sites for two of these Rabs are also present on two coiled-coil proteins of the cis-Golgi, the Drosophila melanogaster orthologues of GM130 and GMAP-210. We suggest an integrated model for a tentacular Golgi in which coiled-coil proteins surround the Golgi to capture and retain Rab-containing membranes, excluding other structures such as ribosomes. Binding sites for diverse Rabs could ensure that incoming carriers are captured on first contact and moved to their correct destination within the stack.     

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.     

A cryptic Rab1-binding site in the p115 tethering protein

Small GTPases and coiled-coil proteins of the golgin family help to tether COPI vesicles to Golgi membranes. At the cis-side of the Golgi, the Rab1 GTPase binds directly to each of three coiled-coil proteins: p115, GM130, and as now shown, Giantin. Rab1 binds to a coiled-coil region within the tail domain of p115 and this binding is inhibited by the C-terminal, acidic domain of p115. Furthermore, GM130 and Giantin bind to the acidic domain of p115 and stimulate p115 binding to Rab1, suggesting that p115 binding to Rab1 is regulated. Regulation of this interaction by proteins such as GM130 and Giantin may control the membrane recruitment of p115 by Rab1.     

Membrane traffic: a glitch in the Golgi matrix

Golgins are coiled-coil proteins thought to form a matrix important for shaping and organising Golgi cisternae and directing long-range recognition events in vesicular transport. This model is brought into question by new evidence that two golgins, GM130 and golgin-84, contribute to but are not essential for protein transport and Golgi structure.     

A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins

In recent years, a large number of coiled-coil proteins localised to the Golgi apparatus have been identified using antisera from human patients with a variety of autoimmune conditions [1]. Because of their common method of discovery and extensive regions of coiled-coil, they have been classified as a family of proteins, the golgins [1]. This family includes golgin-230/245/256, golgin-97, GM130/golgin-95, golgin-160/MEA-2/GCP170, giantin/macrogolgin and a related group of proteins - possibly splice variants - GCP372 and GCP364[2][3][4][5][6][7][8][9][10][11]. GM130 and giantin have been shown to function in the p115-mediated docking of vesicles with Golgi cisternae [12]. In this process, p115, another coiled-coil protein, is though to bind to giantin on vesicles and to GM130 on cisternae, thus acting as a tether holding the two together [12] [13]. Apart from giantin and GM130, none of the golgins has yet been assigned a function in the Golgi apparatus. In order to obtain clues as to the functions of the golgins, the targeting to the Golgi apparatus of two members of this family, golgin-230/245/256 and golgin-97, was investigated. Each of these proteins was shown to target to the Golgi apparatus through a carboxy-terminal domain containing a conserved tyrosine residue, which was critical for targeting. The domain preferentially bound to Rab6 on protein blots, and mutations that abolished Golgi targeting resulted in a loss of this interaction. Sequence analysis revealed that a family of coiled-coil proteins from mammals, worms and yeast contain this domain at their carboxyl termini. One of these proteins, yeast Imh1p, has previously been shown to have a tight genetic interaction with Rab6 [14]. On the basis of these data, it is proposed that this family of coiled-coil proteins functions in Rab6-regulated membrane-tethering events.     

Localization and domain characterization of Arabidopsis golgin candidates

Golgins are large coiled-coil proteins that play a role in tethering of vesicles to Golgi membranes and in maintaining the overall structure of the Golgi apparatus. Six Arabidopsis proteins with the structural characteristics of golgins were isolated and shown to locate to Golgi stacks when fused to GFP. Two of these golgin candidates (GC1 and GC2) possess C-terminal transmembrane (TM) domains with similarity to the TM domain of human golgin-84. The C-termini of two others (GC3/GDAP1 and GC4) contain conserved GRAB and GA1 domains that are also found in yeast Rud3p and human GMAP210. GC5 shares similarity with yeast Sgm1p and human TMF and GC6 with yeast Uso1p and human p115. When fused to GFP, the C-terminal domains of AtCASP and GC1 to GC6 localized to the Golgi, showing that they contain Golgi localization motifs. The N-termini, on the other hand, label the cytosol or nucleus. Immuno-gold labelling and co-expression with the cis Golgi Q-SNARE Memb11 resulted in a more detailed picture of the sub-Golgi location of some of these putative golgins. Using two independent assays it is further demonstrated that the interaction between GC5, the TMF homologue, and the Rab6 homologues is conserved in plants.     

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.     

Structural basis for Arl1-dependent targeting of homodimeric GRIP domains to the Golgi apparatus

Golgins are large coiled-coil proteins that play a role in Golgi structure and vesicle traffic. The Arf-like GTPase Arl1 regulates the translocation of GRIP domain-containing golgins to Golgi membranes. We report here the 1.7 A resolution structure of human Arl1-GTP in a complex with the GRIP domain of golgin-245. The structure reveals that the GRIP domain consists of an S-shaped arrangement of three helices. The domain forms a homodimer that binds two Arl1-GTPs using two helices from each monomer. The structure is consistent with golgin-245 forming parallel coiled-coils and suggests how Arl1-GTP/GRIP complexes interact with Golgi membranes via the N termini of Arl1-GTP and the C-terminal tails of the GRIP domains. In cells, bivalent association with Arl1-GTP would increase residence time of the golgins on Golgi membranes. Despite no conservation of sequence, topology, or even helical direction, several other effectors form similar interactions with small GTPases via a pair of alpha helices, suggesting a common structural basis for effector recognition.     

Golgi Disruption and Early Embryonic Lethality in Mice Lacking USO1

Golgins are a family of long rod-like proteins characterized by the presence of central coiled-coil domains. Members of the golgin family have important roles in membrane trafficking, where they function as tethering factors that capture transport vesicles and facilitate membrane fusion. Golgin family members also have essential roles in maintaining the organization of the Golgi apparatus. Knockdown of individual golgins in cultured cells resulted in the disruption of the Golgi structure and the dispersal of Golgi marker proteins throughout the cytoplasm. However, these cellular phenotypes have not always been recapitulated in vivo. For example, embryonic development proceeds much further than expected and Golgi disruption was observed in only a subset of cell types in mice lacking the ubiquitously expressed golgin GMAP-210. Cell-type specific functional compensation among golgins may explain the absence of global cell lethality when a ubiquitously expressed golgin is missing. In this study we show that functional compensation does not occur for the golgin USO1. Mice lacking this ubiquitously expressed protein exhibit disruption of Golgi structure and early embryonic lethality, indicating that USO1 is indispensable for early embryonic development.     

In situ cleavage of the acidic domain from the p115 tether inhibits exocytic transport

Golgins are coiled-coil proteins involved in Golgi architecture and function. A complex of golgins (p115, GM130 and giantin), together with the rab1 guanosine triphosphatase and cis Golgi SNAREs, helps to mediate fusion processes at the entry face of the Golgi apparatus. The C-terminal acidic domain of p115 binds specifically to GM130 and giantin. However, deletion of this domain in vivo appears to have no effect on exocytic transport when using an RNA interference depletion/rescue approach (Puthenveedu MA, Linstedt AD. Gene replacement reveals that p115/SNARE interactions are essential for Golgi biogenesis. Proc Natl Acad Sci U S A 2004;101:1253–1256). In this study, we have used a different approach introducing a tobacco etch virus (tev) protease cleavage site into p115 so that the C-terminal domain can be rapidly and specifically released in vivo by microinjection of the tev protease. The results show that cleavage inhibits exocytic transport to the cell surface.     

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.     

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.     

Sequential tethering of Golgins and catalysis of SNAREpin assembly by the vesicle-tethering protein p115

p115 tethers coat protein (COP)I vesicles to Golgi membranes. The acidic COOH-terminal domain of p115 links the Golgins, Giantin on COPI vesicles, to GM130 on Golgi membranes. We now show that a SNARE motif-related domain within p115 stimulates the specific assembly of endogenous Golgi SNAREpins containing the t-SNARE, syntaxin 5. p115 catalyzes the construction of a cognate GOS-28-syntaxin-5 (v-/t-SNARE) complex by first linking the SNAREs to promote their direct interaction. These events are essential for NSF-catalyzed reassembly of postmitotic Golgi vesicles and tubules into mature cisternae. Staging experiments reveal that the linking of Golgins precedes SNAREpin assembly. Thus, p115 coordinates sequential tethering and docking of COPI vesicles by first using long tethers (Golgins) and then short tethers (SNAREs).     

The Golgin Tether Giantin Regulates the Secretory Pathway by Controlling Stack Organization within Golgi Apparatus

Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.     

Golgi tethering factors

Transport of cargo to, through and from the Golgi complex is mediated by vesicular carriers and transient tubular connections. In this review, we describe vesicle tethering events with the understanding that similar events occur during transport via larger structures. Tethering factors can be generally divided into a group of coiled-coil proteins and a group of multi-subunit complexes. Current evidence suggests that these factors function in a variety of membrane-membrane tethering events at the Golgi complex, interact with SNARE molecules, and are regulated by small GTPases of the Rab and Arl families.     

Identification and characterization of AtCASP,a plant transmembrane Golgi matrix protein

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.†These authors have contributed equally to the work     

The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells

Golgi-associated long coiled-coil proteins, often referred to as golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1 (neuroendocrine long coiled-coil protein 1). The present study shows that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K+-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture, but enhanced K+-stimulated hormone secretion in PC12 cells. In all, the results of the present study identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells.     

Golgin-84 is a rab1 binding partner involved in Golgi structure

Members of the golgin family of coiled-coil proteins have been implicated in the tethering of vesicles to Golgi membranes and cisternal membranes to each other. Many also bind to rab GTPases. Golgin-84 is a membrane-anchored golgin that we now show binds preferentially to the GTP form of the rab1 GTPase. It is also present throughout the Golgi stack by immuno-EM. Antibodies to golgin-84 inhibit stacking of cisternal membranes in a cell-free assay for Golgi reassembly, whereas the cytoplasmic domain of golgin-84 stimulates stacking and increases the length of re-assembled stacks. Transient expression of golgin-84 in NRK cells helps prevent the disassembly of the Golgi apparatus normally triggered by treatment with brefeldin A. Together these data suggest that golgin-84 is involved in generating and maintaining the architecture of the Golgi apparatus.