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.     

GCC185 plays independent roles in Golgi structure maintenance and AP-1-mediated vesicle tethering

GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)-containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.     

Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization

Homotypic membrane tethering by the Golgi reassembly and stacking proteins (GRASPs) is required for the lateral linkage of mammalian Golgi ministacks into a ribbon-like membrane network. Although GRASP65 and GRASP55 are specifically localized to cis and medial/trans cisternae, respectively, it is unknown whether each GRASP mediates cisternae-specific tethering and whether such specificity is necessary for Golgi compartmentalization. Here each GRASP was tagged with KillerRed (KR), expressed in HeLa cells, and inhibited by 1-min exposure to light. Significantly, inactivation of either GRASP unlinked the Golgi ribbon, and the immediate effect of GRASP65-KR inactivation was a loss of cis- rather than trans-Golgi integrity, whereas inactivation of GRASP55-KR first affected the trans- and not the cis-Golgi. Thus each GRASP appears to play a direct and cisternae-specific role in linking ministacks into a continuous membrane network. To test the consequence of loss of cisternae-specific tethering, we generated Golgi membranes with a single GRASP on all cisternae. Remarkably, the membranes exhibited the full connectivity of wild-type Golgi ribbons but were decompartmentalized and defective in glycan processing. Thus the GRASP isoforms specifically link analogous cisternae to ensure Golgi compartmentalization and proper processing.     

An update on transport vesicle tethering

Membrane trafficking involves the collection of cargo into nascent transport vesicles that bud off from a donor compartment, translocate along cytoskeletal tracks, and then dock and fuse with their target membranes. Docking and fusion involve initial interaction at a distance (tethering), followed by a closer interaction that leads to pairing of vesicle SNARE proteins (v-SNAREs) with target membrane SNAREs (t-SNAREs), thereby catalyzing vesicle fusion. When tethering cannot take place, transport vesicles accumulate in the cytoplasm. Tethering is generally carried out by two broad classes of molecules: extended, coiled-coil proteins such as the so-called Golgin proteins, or multi-subunit complexes such as the Exocyst, COG or Dsl complexes. This review will focus on the most recent advances in terms of our understanding of the mechanism by which tethers carry out their roles, and new structural insights into tethering complex transactions.     

An update on transport vesicle tethering

Abstract

Membrane trafficking involves the collection of cargo into nascent transport vesicles that bud off from a donor compartment, translocate along cytoskeletal tracks, and then dock and fuse with their target membranes. Docking and fusion involve initial interaction at a distance (tethering), followed by a closer interaction that leads to pairing of vesicle SNARE proteins (v-SNAREs) with target membrane SNAREs (t-SNAREs), thereby catalyzing vesicle fusion. When tethering cannot take place, transport vesicles accumulate in the cytoplasm. Tethering is generally carried out by two broad classes of molecules: extended, coiled-coil proteins such as the so-called Golgin proteins, or multi-subunit complexes such as the Exocyst, COG or Dsl complexes. This review will focus on the most recent advances in terms of our understanding of the mechanism by which tethers carry out their roles, and new structural insights into tethering complex transactions.     

The Rab GTPase Ypt1p and tethering factors couple protein sorting at the ER to vesicle targeting to the Golgi apparatus

GPI-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event can be reproduced in vitro. When extracts from a uso1 mutant were used, the sorting of GPI-anchored proteins from other secretory proteins was defective. Complementation with purified Uso1p restored sorting. The Rab GTPase Ypt1p and the tethering factors Sec34p and Sec35p, but not Bet3p, a member of the TRAPP complex, were also required for protein sorting upon ER exit. Therefore, the Ypt1p tethering complex couples protein sorting in the ER to vesicle targeting to the Golgi apparatus. Sorting of GPI-anchored proteins from other secretory proteins was also observed in vivo. The sorting defect observed in vitro with uso1 and ypt1 mutants was reproduced in vivo.     

Transaldolase: From biochemistry to human disease

The role of the enzyme transaldolase (TAL) in central metabolism, its biochemical properties, structure, and role in human disease is reviewed. The nearly ubiquitous enzyme transaldolase is a part of the pentose phosphate pathway and transfers a dihydroxyacetone group from donor compounds (fructose 6-phosphate or sedoheptulose 7-phosphate) to aldehyde acceptor compounds. The phylogeny of transaldolases shows that five subfamilies can be distinguished, three of them with proven TAL enzyme activity, one with unclear function, and the fifth subfamily comprises transaldolase-related enzymes, the recently discovered fructose 6-phosphate aldolases. The three-dimensional structure of a bacterial (Escherichia coli TAL B) and the human enzyme (TALDO1) has been solved. Based on the 3D-structure and mutagenesis studies, the reaction mechanism was deduced. The cofactor-less enzyme proceeds with a Schiff base intermediate (bound dihydroxyacetone). While a transaldolase deficiency is well tolerated in many microorganisms, it leads to severe symptoms in homozygous TAL-deficient human patients. The involvement of TAL in oxidative stress and apoptosis, in multiple sclerosis, and in cancer is discussed.     

Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.     

Retrograde vesicle transport in the Golgi

The Golgi apparatus is the central sorting and biosynthesis hub of the secretory pathway, and uses vesicle transport for the recycling of its resident enzymes. This system must operate with high fidelity and efficiency for the correct modification of secretory glycoconjugates. In this review, we discuss recent advances on how coats, tethers, Rabs and SNAREs cooperate at the Golgi to achieve vesicle transport. We cover the well understood vesicle formation process orchestrated by the COPI coat, and the comprehensively documented fusion process governed by a set of Golgi localised SNAREs. Much less clear are the steps in-between formation and fusion of vesicles, and we therefore provide a much needed update of the latest findings about vesicle tethering. The interplay between Rab GTPases, golgin family coiled-coil tethers and the conserved oligomeric Golgi (COG) complex at the Golgi are thoroughly evaluated.     

Role of vesicle tethering factors in the ER-Golgi membrane traffic

Tethers are a diverse group of loosely related proteins and protein complexes grouped into three families based on structural and functional similarities. A well-accepted role for tethering factors is the initial attachment of transport carriers to acceptor membranes prior to fusion. However, accumulating evidence indicates that tethers are more than static bridges. Tethers have been shown to interact with components of the fusion machinery and with components involved in vesicle formation. Tethers belonging to the three families act at the same stage of traffic, suggesting that they mediate distinct events during vesicle tethering. Thus, multiple tether-facilitated events are required to provide selectivity to vesicle fusion. In this review, we highlight findings that support this model.     

Arabidopsis myosin XIK interacts with the exocyst complex to facilitate vesicle tethering during exocytosis

Myosin motors are essential players in secretory vesicle trafficking and exocytosis in yeast and mammalian cells; however, similar roles in plants remain a matter for debate, at least for diffusely growing cells. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) myosin XIK, via its globular tail domain (GTD), participates in the vesicle tethering step of exocytosis through direct interactions with the exocyst complex. Specifically, myosin XIK GTD bound directly to several exocyst subunits in vitro and functional fluorescently tagged XIK colocalized with multiple exocyst subunits at plasma membrane (PM)-associated stationary foci. Moreover, genetic and pharmacological inhibition of myosin XI activity reduced the rate of appearance and lifetime of stationary exocyst complexes at the PM. By tracking single exocytosis events of cellulose synthase (CESA) complexes with high spatiotemporal resolution imaging and pair-wise colocalization of myosin XIK, exocyst subunits, and CESA6, we demonstrated that XIK associates with secretory vesicles earlier than exocyst and is required for the efficient localization and normal dynamic behavior of exocyst complex at the PM tethering site. This study reveals an important functional role for myosin XI in secretion and provides insights about the dynamic regulation of exocytosis in plants.     

Allosteric regulation of GRASP protein-dependent Golgi membrane tethering by mitotic phosphorylation

Mitotic phosphorylation of the conserved GRASP domain of GRASP65 disrupts its self-association, leading to a loss of Golgi membrane tethering, cisternal unlinking, and Golgi breakdown. Recently, the structural basis of the GRASP self-interaction was determined, yet the mechanism by which phosphorylation disrupts this activity is unknown. Here, we present the crystal structure of a GRASP phosphomimic containing an aspartic acid substitution for a serine residue (Ser-189) that in GRASP65 is phosphorylated by PLK1, causing a block in membrane tethering and Golgi ribbon formation. The structure revealed a conformational change in the GRASP internal ligand that prevented its insertion into the PDZ binding pocket, and gel filtration assays showed that this phosphomimic mutant exhibited a significant reduction in dimer formation. Interestingly, the structure also revealed an apparent propagation of conformational change from the site of phosphorylation to the shifted ligand, and alanine substitution of two residues (Glu-145 and Ser-146) at penultimate positions in this chain rescued dimer formation by the phosphomimic. These data reveal the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation.     

The binary interacting network of the conserved oligomeric Golgi tethering complex

Several recent studies have revealed the existence of a conserved oligomeric Golgi (COG) complex consisting of several novel proteins as well as known Golgi proteins that were identified by independent approaches. The mammalian COG complex contains eight subunits: COG1/LdlBp, COG2/LdlCp, COG3/Sec34, COG4/Cod1, COG5/GTC-90/Cod4, COG6/Cod2, COG7, and COG8/Dor1. COG1, COG2, and COG7 seem structurally unique to mammalian cells, whereas the other five subunits are structurally conserved in yeast, which also contains three other unique proteins (COG1/Sec36p/Cod3p, COG2/Sec35p, and COG7/Cod5p). We report here the network of intermolecular interactions of the COG complex, revealed by in vitro translation and co-immunoprecipitation approaches. Our results suggest that COG4 serves as a core component of the complex by interacting directly with COG1, COG2, COG5, and COG7. COG3 is incorporated by its direct interaction with COG1 and COG2, whereas COG6 and COG8 do not interact with any individual subunit. Incorporation of COG6 into the complex depends on the concerted interaction of both COG5 and COG7, whereas optimal incorporation of COG8 depends on the concerted interaction of COG5, COG6, and COG7. Because COG4 (together with COG1, COG2, and COG3) is among the four essential genes of the COG complex in yeast, this molecular network highlights the structural basis for a crucial role of COG4 in the assembly/function of the complex. A model for the assembly of the COG complex is presented.     

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.     

Structures and mechanisms of vesicle coat components and multisubunit tethering complexes

Eukaryotic cells face a logistical challenge in ensuring prompt and precise delivery of vesicular cargo to specific organelles within the cell. Coat protein complexes select cargo and initiate vesicle formation, while multisubunit tethering complexes participate in the delivery of vesicles to target membranes. Understanding these macromolecular assemblies has greatly benefited from their structural characterization. Recent structural data highlight principles in coat recruitment and uncoating in both the endocytic and retrograde pathways, and studies on the architecture of tethering complexes provide a framework for how they might link vesicles to the respective acceptor compartments and the fusion machinery.     

GFP-golvesin constructs to study Golgi tubulation and post-Golgi vesicle dynamics in phagocytosis

Dictyostelium cells are professional phagocytes that are optimally suited for the imaging of phagosome processing from particle uptake to exocytosis. In order to design fluorescent probes for monitoring membrane trafficking in the endocytic pathway, we have dissected a membrane protein, golvesin, and have linked fragments of its sequence to GFP. Endogenous golvesin is partitioned between the ER, the Golgi apparatus, endosomes, and the contractile vacuole complex. We have localized signals that are required for exit from the Golgi to post-Golgi compartments to the C-terminal region of the golvesin sequence. One GFP-tagged fragment turned out to be a highly specific Golgi marker and was used to demonstrate the interaction of Golgi tubules with phagosomes. Signals essential for the retrieval of golvesin at the end of phagosome processing were localized to the N-terminal region. A truncated golvesin construct escaping retrieval was employed in recording the delivery of a phagosomal protein to the plasma membrane. Applying this construct to a phagosome filled with multiple particles, we observed that the phagosome is segmented during exocytosis, meaning that sequential release of particles alternates with membrane fusion.