The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins.

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

Role of GARP Vesicle Tethering Complex in Golgi Physiology.

The Golgi associated retrograde protein complex (GARP) is an evolutionarily conserved component of Golgi membrane trafficking machinery that belongs to the Complexes Associated with Tethering Containing Helical Rods (CATCHR) family. Like other multisubunit tethering complexes such as COG, Dsl1, and Exocyst, the GARP is believed to function by tethering and promoting fusion of the endosome-derived small trafficking intermediate. However, even twenty years after its discovery, the exact structure and the functions of GARP are still an enigma. Recent studies revealed novel roles for GARP in Golgi physiology and identified human patients with mutations in GARP subunits. In this review, we summarized our knowledge of the structure of the GARP complex, its protein partners, GARP functions related to Golgi physiology, as well as cellular defects associated with the dysfunction of GARP subunits.

Modeling the Cryo-EM Structure of the Exocyst Complex.

Multi-subunit tethering complexes (MTCs) are a family of evolutionarily conserved large protein complexes that function to tether intracellular vesicles from the donor compartments to the membrane of receptor compartments. The exocyst complex is an octameric MTC that tethers the post-Golgi secretory vesicles to the plasma membrane. To learn the function and regulation of the exocyst complex, it is crucial to understand the structure of the complex. We have solved the cryo-EM structure of the exocyst complex at 4.4 Angstrom (Å) resolution and detected the spatial relationship between the eight subunits using chemical cross-linking mass spectrometry. Here, we describe the method of modeling and validating the cryo-EM structure of the exocyst complex. This method could provide a guide for modeling of other protein complexes of which the structures are solved at medium to near-atomic resolution.

Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines.

The Golgi-associated retrograde protein (GARP) complex is proposed to tether endosome-derived transport vesicles, but the exact function and mechanism of GARP action are not completely understood. To uncover the GARP function in human cells, we employ CRISPR/Cas9 strategy and knock out (KO) the unique VPS54 subunit of the GARP complex. In this chapter, we describe the detailed method of generating CRISPR/Cas9-mediated VPS54-KO in hTERT-RPE1 cells, rescue of resulting KO cells with stable near-endogenous expression of myc-tagged VPS54, and validation of KO and rescued (KO-R) cells using Western blot and immunofluorescence approaches. This approach is helpful in uncovering new functions of the GARP and other vesicle tethering complexes.

Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System.

Conserved oligomeric Golgi (COG) complex orchestrates intra-Golgi retrograde trafficking and glycosylation of macromolecules, but the detailed mechanism of COG action is unknown. Previous studies employed prolonged protein knockout and knockdown approaches which may potentially generate off-target and indirect mutant phenotypes. To achieve a fast depletion of COG subunits in human cells, the auxin-inducible degradation system was employed. This method of protein regulation allows a very fast and efficient depletion of COG subunits, which provides the ability to accumulate COG complex dependent (CCD) vesicles and investigate initial cellular defects associated with the acute depletion of COG complex subunits. This protocol is applicable to other vesicle tethering complexes and can be utilized to investigate anterograde and retrograde intracellular membrane trafficking pathways.

Determine the Function of the Exocyst in Vesicle Tethering by Ectopic Targeting.

We describe an assay, in which ectopically targeting the exocyst subunit Sec3 to mitochondria is used to determine its role in tethering of post-Golgi vesicles to the plasma membrane. In the assay, we use a plasmid that encodes a fusion protein of the mitochondria protein Tom20 and Sec3 N-terminally tagged with the florescence protein mCherry, and coexpress the plasmid in yeast cells with CIT1-GFP, a marker protein of mitochondria. We then detect the colocalization between Sec3 and CIT1 and other exocyst subunits such as Sec5 on mitochondria using fluorescence microscopy. We further detect the colocalization between Sec3 and Sec4, a Rab protein and a marker of post-Golgi vesicles. Through this assay, we propose that the exocyst subunit Sec3 recruits the other exocyst subunits and secretory vesicles to a target membrane, suggesting that it plays a pivotal role in vesicle tethering. This approach is likely appropriate for studying other tethering complexes at their specific stages of trafficking and may also be used in other eukaryotic cells such as the cultured mammalian cells.

Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases.

Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogeneous, and patients primarily represent neurological, skeletal, and hepatic abnormalities. The establishment of a cellular COG disease model will benefit the molecular study of the disease, explaining the detailed sequence of the interplay between the COG complex and the trafficking machinery. Moreover, patient fibroblasts are not a good representative of all the organ systems and cell types that are affected by COG mutations. We developed and characterized cellular models for human COG4 mutations, specifically in RPE1 and HEK293T cell lines. Using a combination of CRISPR/Cas9 and lentiviral transduction technologies, both myc-tagged wild-type and mutant (G516R and R729W) COG4 proteins were expressed under the endogenous COG4 promoter. Constructed isogenic cell lines were comprehensively characterized using biochemical, microscopy (superresolution and electron), and proteomics approaches. The analysis revealed similar stability and localization of COG complex subunits, wild-type cell growth, and normal Golgi morphology in all three cell lines. Importantly, COG4-G516R cells demonstrated increased HPA-647 binding to the plasma membrane glycoconjugates, while COG4-R729W cells revealed high GNL-647 binding, indicating specific defects in O- and N-glycosylation. Both mutant cell lines express an elevated level of heparin sulfate proteoglycans. Moreover, a quantitative mass-spectrometry analysis of proteins secreted by COG-deficient cell lines revealed abnormal secretion of SIL1 and ERGIC-53 proteins by COG4-G516R cells. Interestingly, the clinical phenotype of patients with congenital mutations in the SIL1 gene (Marinesco-Sjogren syndrome) overlaps with the phenotype of COG4-G516R patients (Saul-Wilson syndrome). Our work is the first compressive study involving the creation of different COG mutations in different cell lines other than the patient's fibroblast. It may help to address the underlying cause of the phenotypic defects leading to the discovery of a proper treatment guideline for COG-CDGs.

Golgi inCOGnito: From vesicle tethering to human disease.

The Conserved Oligomeric Golgi (COG) complex, a multi-subunit vesicle tethering complex of the CATCHR (Complexes Associated with Tethering Containing Helical Rods) family, controls several aspects of cellular homeostasis by orchestrating retrograde vesicle traffic within the Golgi. The COG complex interacts with all key players regulating intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, and vesicular coats. In cells, COG deficiencies result in the accumulation of non-tethered COG-complex dependent (CCD) vesicles, dramatic morphological and functional abnormalities of the Golgi and endosomes, severe defects in N- and O- glycosylation, Golgi retrograde trafficking, sorting and protein secretion. In humans, COG mutations lead to severe multi-systemic diseases known as COG-Congenital Disorders of Glycosylation (COG-CDG). In this report, we review the current knowledge of the COG complex and analyze COG-related trafficking and glycosylation defects in COG-CDG patients.

Autophagosome-Lysosome Fusion.

Macroautophagy is a conserved catabolic process observed in all eukaryotic cells, during which selected cellular components are transported to and broken down within lysosomes. The process starts with the capture of unnecessary material into autophagosomes, which is followed by autophagosome-lysosome fusion to generate autolysosomes that degrade the cargo. In the past quarter-century, our knowledge about autophagosome formation almost exponentially increased, while the later steps were much less studied. This fortunately changed in the past few years, with more and more publications focusing on the fate of the completed autophagosome. In this review, we aspire to summarize the current knowledge about the molecular mechanisms of autophagosome-lysosome fusion.

Nanomachinery Organizing Release at Neuronal and Ribbon Synapses.

A critical aim in neuroscience is to obtain a comprehensive view of how regulated neurotransmission is achieved. Our current understanding of synapses relies mainly on data from electrophysiological recordings, imaging, and molecular biology. Based on these methodologies, proteins involved in a synaptic vesicle (SV) formation, mobility, and fusion at the active zone (AZ) membrane have been identified. In the last decade, electron tomography (ET) combined with a rapid freezing immobilization of neuronal samples opened a window for understanding the structural machinery with the highest spatial resolution in situ. ET provides significant insights into the molecular architecture of the AZ and the organelles within the presynaptic nerve terminal. The specialized sensory ribbon synapses exhibit a distinct architecture from neuronal synapses due to the presence of the electron-dense synaptic ribbon. However, both synapse types share the filamentous structures, also commonly termed as tethers that are proposed to contribute to different steps of SV recruitment and exocytosis. In this review, we discuss the emerging views on the role of filamentous structures in SV exocytosis gained from ultrastructural studies of excitatory, mainly central neuronal compared to ribbon-type synapses with a focus on inner hair cell (IHC) ribbon synapses. Moreover, we will speculate on the molecular entities that may be involved in filament formation and hence play a crucial role in the SV cycle.

Vesicle sub-pool organization at inner hair cell ribbon synapses.

The afferent inner hair cell synapse harbors the synaptic ribbon, which ensures a constant vesicle supply. Synaptic vesicles (SVs) are arranged in morphologically discernable pools, linked via filaments to the ribbon or the presynaptic membrane. We propose that filaments play a major role in SV resupply and exocytosis at the ribbon. Using advanced electron microscopy, we demonstrate that SVs are organized in sub-pools defined by the filament number per vesicle and its connections. Upon stimulation, SVs increasingly linked to other vesicles and to the ribbon, whereas single-tethered SVs dominated at the membrane. Mutant mice for the hair cell protein otoferlin (pachanga, OtofPga/Pga ) are profoundly deaf with reduced sustained release, serving as a model to investigate the SV replenishment at IHCs. Upon stimulation, multiple-tethered and docked vesicles (rarely observed in wild-type) accumulated at OtofPga/Pga active zones due to an impairment downstream of docking. Conclusively, vesicles are organized in sub-pools at ribbon-type active zones by filaments to support vesicle supply, transport, and finally release.

A Presynaptic Liquid Phase Unlocks the Vesicle Cluster.

How are synaptic vesicles tied together in a nerve terminal? A recent study by Milovanovic and colleagues offers a new mechanism for this old and important problem: synapsin proteins establish a liquid phase that clusters vesicles. Liquid-liquid phase separation provides a fluid-like state that accommodates the dynamic demands of presynaptic vesicle traffic.

Emerging roles of cortical microtubule-membrane interactions.

Plant cortical microtubules have crucial roles in cell wall development. Cortical microtubules are tightly anchored to the plasma membrane in a highly ordered array, which directs the deposition of cellulose microfibrils by guiding the movement of the cellulose synthase complex. Cortical microtubules also interact with several endomembrane systems to regulate cell wall development and other cellular events. Recent studies have identified new factors that mediate interactions between cortical microtubules and endomembrane systems including the plasma membrane, endosome, exocytic vesicles, and endoplasmic reticulum. These studies revealed that cortical microtubule-membrane interactions are highly dynamic, with specialized roles in developmental and environmental signaling pathways. A recent reconstructive study identified a novel function of the cortical microtubule-plasma membrane interaction, which acts as a lateral fence that defines plasma membrane domains. This review summarizes recent advances in our understanding of the mechanisms and functions of cortical microtubule-membrane interactions.

Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay.

Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP. Given that GalT is recycled to the cisterna where it is located at steady state, GalT-containing vesicles target GalT-containing cisternal membranes. Golgi membranes were therefore isolated from GalT-CFP expressing cells, while vesicles were prepared from GalT-YFP expressing ones. Incubating CFP-labelled Golgi with YFP-labelled vesicles in the presence of cytosol and an energy regeneration mixture at 37 °C produced a significant increase in CFP-YFP co-localization upon fluorescent imaging of the mixture compared to incubation on ice. The assay was validated to require energy, proteins and physiologically important trafficking components such as Rab GTPases and the conserved oligomeric Golgi tethering complex. This assay is useful for the investigation of both physiological and pathological changes that affect the Golgi trafficking machinery, in particular, vesicle tethering.

The cell polarity proteins Boi1p and Boi2p stimulate vesicle fusion at the plasma membrane of yeast cells.

Eukaryotic cells can direct secretion to defined regions of their plasma membrane. These regions are distinguished by an elaborate architecture of proteins and lipids that are specialized to capture and fuse post-Golgi vesicles. Here, we show that the proteins Boi1p and Boi2p are important elements of this area of active exocytosis at the tip of growing yeast cells. Cells lacking Boi1p and Boi2p accumulate secretory vesicles in their buds. The essential PH domains of Boi1p and Boi2p interact with Sec1p, a protein required for SNARE complex formation and vesicle fusion. Sec1p loses its tip localization in cells depleted of Boi1p and Boi2p but overexpression of Sec1p can partially compensate for their loss. The capacity to simultaneously bind phospholipids, Sec1p, multiple subunits of the exocyst, Cdc42p and the module for generating active Cdc42p identify Boi1p and Boi2p as essential mediators between exocytosis and polar growth.

Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses.

RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca2+ channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1ß are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM-Munc13-RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis.SIGNIFICANCE STATEMENT RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca2+ channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.

The golgin coiled-coil proteins capture different types of transport carriers via distinct N-terminal motifs.

BACKGROUND: The internal organization of cells depends on mechanisms to ensure that transport carriers, such as vesicles, fuse only with the correct destination organelle. Several types of proteins have been proposed to confer specificity to this process, and we have recently shown that a set of coiled-coil proteins on the Golgi, called golgins, are able to capture specific classes of carriers when relocated to an ectopic location. RESULTS: Mapping of six different golgins reveals that, in each case, a short 20-50 residue region is necessary and sufficient to capture specific carriers. In all six of GMAP-210, golgin-84, TMF, golgin-97, golgin-245, and GCC88, this region is located at the extreme N-terminus of the protein. The vesicle-capturing regions of GMAP-210, golgin-84, and TMF capture intra-Golgi vesicles and share some sequence features, suggesting that they act in a related, if distinct, manner. In the case of GMAP-210, this shared feature is in addition to a previously characterized "amphipathic lipid-packing sensor" motif that can capture highly curved membranes, with the two motifs being apparently involved in capturing distinct types of vesicles. Of the three GRIP domain golgins that capture endosome-to-Golgi carriers, golgin-97 and golgin-245 share a closely related capture motif, whereas that in GCC88 is distinct, suggesting that it works by a different mechanism and raising the possibility that the three golgins capture different classes of endosome-derived carriers that share many cargos but have distinct features for recognition at the Golgi. CONCLUSIONS: For six different golgins, the capture of carriers is mediated by a short region at the N-terminus of the protein. There appear to be at least four different types of motif, consistent with specific golgins capturing specific classes of carrier and implying the existence of distinct receptors present on each of these different carrier classes.

ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking.

COPI-coated vesicles mediate retrograde membrane traffic from the cis-Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER-resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V-dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI•Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.

COG Complex Complexities: Detailed Characterization of a Complete Set of HEK293T Cells Lacking Individual COG Subunits.

The Conserved Oligomeric Golgi complex is an evolutionarily conserved multisubunit tethering complex (MTC) that is crucial for intracellular membrane trafficking and Golgi homeostasis. The COG complex interacts with core vesicle docking and fusion machinery at the Golgi; however, its exact mechanism of action is still an enigma. Previous studies of COG complex were limited to the use of CDGII (Congenital disorders of glycosylation type II)-COG patient fibroblasts, siRNA mediated knockdowns, or protein relocalization approaches. In this study we have used the CRISPR approach to generate HEK293T knock-out (KO) cell lines missing individual COG subunits. These cell lines were characterized for glycosylation and trafficking defects, cell proliferation rates, stability of COG subunits, localization of Golgi markers, changes in Golgi structure, and N-glycan profiling. We found that all KO cell lines were uniformly deficient in cis/medial-Golgi glycosylation and each had nearly abolished binding of Cholera toxin. In addition, all cell lines showed defects in Golgi morphology, retrograde trafficking and sorting, sialylation and fucosylation, but severities varied according to the affected subunit. Lobe A and Cog6 subunit KOs displayed a more severely distorted Golgi structure, while Cog2, 3, 4, 5, and 7 knock outs had the most hypo glycosylated form of Lamp2. These results led us to conclude that every subunit is essential for COG complex function in Golgi trafficking, though to varying extents. We believe that this study and further analyses of these cells will help further elucidate the roles of individual COG subunits and bring a greater understanding to the class of MTCs as a whole.

Bridging the Gap between Glycosylation and Vesicle Traffic.

Glycosylation is recognized as a vitally important posttranslational modification. The structure of glycans that decorate proteins and lipids is largely dictated by biosynthetic reactions occurring in the Golgi apparatus. This biosynthesis relies on the relative distribution of glycosyltransferases and glycosidases, which is maintained by retrograde vesicle traffic between Golgi cisternae. Tethering of vesicles at the Golgi apparatus prior to fusion is regulated by Rab GTPases, coiled-coil tethers termed golgins and the multisubunit tethering complex known as the conserved oligomeric Golgi (COG) complex. In this review we discuss the mechanisms involved in vesicle tethering at the Golgi apparatus and highlight the importance of tethering in the context of glycan biosynthesis and a set of diseases known as congenital disorders of glycosylation.