Golgi SNARE Research Articles

Syntaxin-5's flexibility in SNARE pairing supports Golgi functions.

Deficiency in the conserved oligomeric Golgi (COG) complex that orchestrates SNARE-mediated tethering/fusion of vesicles that recycle the Golgi's glycosylation machinery results in severe glycosylation defects. Although two major Golgi v-SNAREs, GS28/GOSR1, and GS15/BET1L, are depleted in COG-deficient cells, the complete knockout of GS28 and GS15 only modestly affects Golgi glycosylation, indicating the existence of an adaptation mechanism in Golgi SNARE. Indeed, quantitative mass-spectrometry analysis of STX5-interacting proteins revealed two novel Golgi SNARE complexes-STX5/SNAP29/VAMP7 and STX5/VTI1B/STX8/YKT6. These complexes are present in wild-type cells, but their usage is significantly increased in both GS28- and COG-deficient cells. Upon GS28 deletion, SNAP29 increased its Golgi residency in a STX5-dependent manner. While STX5 depletion and Retro2-induced diversion from the Golgi severely affect protein glycosylation, GS28/SNAP29 and GS28/VTI1B double knockouts alter glycosylation similarly to GS28 KO, indicating that a single STX5-based SNARE complex is sufficient to support Golgi glycosylation. Importantly, co-depletion of three Golgi SNARE complexes in GS28/SNAP29/VTI1B TKO cells resulted in severe glycosylation defects and a reduced capacity for glycosylation enzyme retention at the Golgi. This study demonstrates the remarkable plasticity in SXT5-mediated membrane trafficking, uncovering a novel adaptive response to the failure of canonical intra-Golgi vesicle tethering/fusion machinery.

Diverse Role of SNARE Protein GS28 in Vesicle Trafficking and Diseases.

Golgi SNARE, with a size of 28 kD (GS28), is a transmembrane protein and mainly localizes to the Golgi apparatus. It is considered a core part of the Golgi SNARE complex in the Endoplasmic Reticulum (ER)-Golgi transport and regulates the docking and fusion of transport vesicles effectively. In recent years, increasing studies have indicated that various intracellular transport events are regulated by different GS28-based SNARE complexes. Moreover, GS28 is also involved in numerous functional signaling pathways related to different diseases via interacting with other SNARE proteins and affecting protein maturation and secretion. However, the precise function of GS28 in different disease models and the regulatory network remains unclear. In this review, we mainly provide a concise overview of the function and regulation of GS28 in vesicle trafficking and diseases and summarize the signaling pathways regarding potential mechanisms. Although some critical points about the significance of GS28 in disease treatment still need further investigation, more reliable biotechnical or pharmacological strategies may be developed based on a better understanding of the diverse role of GS28 in vesicle trafficking and other biological processes.

Quantitative Proteomics Analysis of Purified Rat Liver Golgi.

The Golgi is the central organelle in the secretory pathway, essential for post-translational modifications, sorting and trafficking of secretory and membrane proteins and lipids in all eukaryotic cells. During mitosis, the mammalian Golgi membranes undergo continuous disassembly and reassembly processes which are critical for Golgi biogenesis during the cell division. To better understand the underlying molecular mechanism of this highly dynamic process, we analyzed the proteins that are in or associated with interphase and mitotic Golgi membranes using an in vitro Golgi assembly assay and quantitative proteomics. In this study, by combining an isobaric mass tag labeling strategy with OFFGEL peptide fractionation, LC-MS/MS analyses identified and quantified a total of 1193 Golgi-resident or -associated proteins. These proteins included Golgi structural proteins, Golgi-resident enzymes, Rab GTPases, and SNARE proteins. This systematic quantitative proteomic study revealed the comprehensive molecular machinery of the Golgi and the dynamic protein changes in its disassembly and reassembly processes. Here we describe the detailed procedures and protocols for this analysis.

Ubiquitination drives COPI priming and Golgi SNARE localization.

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae. The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of β'-COP or through an unrelated ubiquitin-binding domain is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

CAMLG-CDG: a novel congenital disorder of glycosylation linked to defective membrane trafficking.

The transmembrane domain recognition complex (TRC) pathway is required for the insertion of C-terminal tail-anchored (TA) proteins into the lipid bilayer of specific intracellular organelles such as the endoplasmic reticulum (ER) membrane. In order to facilitate correct insertion, the recognition complex (consisting of BAG6, GET4 and UBL4A) must first bind to TA proteins and then to GET3 (TRC40, ASNA1), which chaperones the protein to the ER membrane. Subsequently, GET1 (WRB) and CAML form a receptor that enables integration of the TA protein within the lipid bilayer. We report an individual with the homozygous c.633 + 4A>G splice variant in CAMLG, encoding CAML. This variant leads to aberrant splicing and lack of functional protein in patient-derived fibroblasts. The patient displays a predominantly neurological phenotype with psychomotor disability, hypotonia, epilepsy and structural brain abnormalities. Biochemically, a combined O-linked and type II N-linked glycosylation defect was found. Mislocalization of syntaxin-5 in patient fibroblasts and in siCAMLG deleted Hela cells confirms this as a consistent cellular marker of TRC dysfunction. Interestingly, the level of the v-SNARE Bet1L is also drastically reduced in both of these models, indicating a fundamental role of the TRC complex in the assembly of Golgi SNARE complexes. It also points towards a possible mechanism behind the hyposialylation of N and O-glycans. This is the first reported patient with pathogenic variants in CAMLG. CAMLG-CDG is the third disorder, after GET4 and GET3 deficiencies, caused by pathogenic variants in a member of the TRC pathway, further expanding this novel group of disorders.

Syntaxin 5 determines Weibel-Palade body size and von Willebrand factor secretion by controlling Golgi architecture.

von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells. VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPB), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde endoplasmic reticulum-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in endothelial cells using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted endothelial cells exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. In brief, we identified SNARE protein STX5 as a novel regulator of WPB biogenesis.

The Parkinson’s Disease Protein LRRK2 Interacts with the GARP Complex to Promote Retrograde Transport to the trans-Golgi Network

SUMMARYMutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease (PD). However, the precise function of LRRK2 remains unclear. We report an interaction between LRRK2 and VPS52, a subunit of the Golgi-associated retrograde protein (GARP) complex that identifies a function of LRRK2 in regulating membrane fusion at the trans-Golgi network (TGN). At the TGN, LRRK2 further interacts with the Golgi SNAREs VAMP4 and Syntaxin-6 and acts as a scaffolding platform that stabilizes the GARP-SNAREs complex formation. Therefore, LRRK2 influences both retrograde and post-Golgi trafficking pathways in a manner dependent on its GTP binding and kinase activity. This action is exaggerated by mutations associated with Parkinson’s disease and can be blocked by kinase inhibitors. Disruption of GARP sensitizes dopamine neurons to mutant LRRK2 toxicity in C. elegans, showing that these pathways are interlinked in vivo and suggesting a link in PD.

A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus.

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the β subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.

Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2.

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes, and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. In contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from endosomes to the Golgi. We thereby identify Sec16A as a druggable target, and provide evidence for a non-SNARE function for syntaxin-5 in interaction with the GPP130.

Regulation of Vesicle Formation at the Golgi Complex by the Arf GEFs Gea1 and Gea2

In eukaryotes, vesicle formation at the Golgi complex is initiated by the conserved small GTPase Arf1. In budding yeast, Arf1 localization and activity is controlled by three separate guanine nucleotide exchange factors (GEFs) Gea1, Gea2, and Sec7. Sec7 activates Arf1 at the trans‐Golgi network, and mechanisms regulating the activity and recruitment of Sec7 to the Golgi have been well characterized. For example, Sec7 is recruited to the TGN by four small GTPases including its substrate Arf1. However, Gea1 and Gea2, which activate Arf1 at earlier Golgi compartments, do not share the same regulatory mechanisms as Sec7. While initially thought to be redundant paralogs, we have found that Gea1 and Gea2 localize to separate regions of the Golgi despite sharing common regulatory elements. As such, the purpose and mechanism for their differential localization remains unclear. I aim to understand the functional relevance for their distinct localization and will present preliminary evidence suggesting that Gea recruitment is coordinated by Golgi SNARE complexes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Golgin protein Coy1 functions in intra-Golgi retrograde transport and interacts with the COG complex and Golgi SNAREs.

Extended coiled-coil proteins of the Golgin family play prominent roles in maintaining the structure and function of the Golgi complex. Here we further investigate the Golgin protein Coy1 and document its function in retrograde transport between early Golgi compartments. Cells that lack Coy1 displayed a reduced half-life of the Och1 mannosyltransferase, an established cargo of intra-Golgi retrograde transport. Combining the coy1Δ mutation with deletions in other putative retrograde Golgins (sgm1Δ and rud3Δ) caused strong glycosylation and growth defects and reduced membrane association of the Conserved Oligomeric Golgi complex. In contrast, overexpression of COY1 inhibited the growth of mutant strains deficient in fusion activity at the Golgi (sed5-1 and sly1-ts). To map Coy1 protein interactions, co-immunoprecipitation experiments revealed an association with the Conserved Oliogmeric Golgi (COG) complex and with intra-Golgi SNARE proteins. These physical interactions are direct, as Coy1 was efficiently captured in vitro by Lobe A of the COG complex and the purified SNARE proteins Gos1, Sed5 and Sft1. Thus, our genetic, in vivo, and biochemical data indicate a role for Coy1 in regulating COG complex-dependent fusion of retrograde-directed COPI vesicles.

Soybean Golgi SNARE 12 protein interacts with Soybean mosaic virus encoded P3N-PIPO protein

Soybean mosaic virus (SMV), a member of the Potyvirus genus, is one of the most prevalent and devastating viral pathogens in soybean-growing regions worldwide. It is generally accepted that symptom development of a viral plant disease results from molecular interactions between the virus and its host plant. P3N-PIPO, as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO of the Potyvirus, plays a key role of viral cell-to-cell movement. This study provides evidence that Golgi SNARE 12 (designated as GOS12) protein of soybean interacts with SMV P3N-PIPO via a two-hybrid yeast system by screening a soybean cDNA library. Bimolecular fluorescence complementation (BiFC) assay further confirmed the interaction, which occurred in the cytomembrane of Nicotiana benthamiana cells. We also confirmed that the domain involved in the interaction is PIPO domain of P3N-PIPO by two-hybrid yeast system and BiFC. It is possible that the GOS12 is an essential host factor for PD targeting of P3N-PIPO protein of potyvirus.

Sec24C/D-isoform-specific sorting of the preassembled ER-Golgi Q-SNARE complex.

Secretory proteins are exported from the endoplasmic reticulum in COPII vesicles. SNARE proteins-core machinery for membrane fusion-are incorporated into COPII vesicles by direct interaction with Sec24. Here we report a novel mechanism for sorting of the ER-Golgi Q-SNAREs into COPII vesicles. Different mammalian Sec24 isoforms recruit either the R-SNARE Sec22b or the Q-SNAREs Syntaxin5, GS27, and Bet1. Syntaxin5 is the only Q-SNARE that directly interacts with Sec24C, requiring its "open" conformation. Mutation within the IxM cargo-binding site of Sec24C led to a drastic reduction in sorting of all three Q-SNAREs into COPII vesicles, implying their ER export as a preassembled complex. Analysis of immunoisolated COPII vesicles and intracellular localization of Sec24 isoforms indicate that all ER-Golgi SNAREs are present on the same vesicle. Combined with existing data, our findings yield a general concept of how Sec24 isoforms can recruit fusogenic SNARE subunits to keep them functionally apart and thus prime mammalian COPII vesicles for homotypic fusion.

Stathmin 1/2-triggered microtubule loss mediates Golgi fragmentation in mutant SOD1 motor neurons.

BackgroundPathological Golgi fragmentation represents a constant pre-clinical feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) but its molecular mechanisms remain hitherto unclear.ResultsHere, we show that the severe Golgi fragmentation in transgenic mutant SOD1G85R and SOD1G93A mouse motor neurons is associated with defective polymerization of Golgi-derived microtubules, loss of the COPI coat subunit β-COP, cytoplasmic dispersion of the Golgi tether GM130, strong accumulation of the ER-Golgi v-SNAREs GS15 and GS28 as well as tubular/vesicular Golgi fragmentation. Data mining, transcriptomic and protein analyses demonstrate that both SOD1 mutants cause early presymptomatic and rapidly progressive up-regulation of the microtubule-destabilizing proteins Stathmins 1 and 2. Remarkably, mutant SOD1-triggered Golgi fragmentation and Golgi SNARE accumulation are recapitulated by Stathmin 1/2 overexpression but completely rescued by Stathmin 1/2 knockdown or the microtubule-stabilizing drug Taxol.ConclusionsWe conclude that Stathmin-triggered microtubule destabilization mediates Golgi fragmentation in mutant SOD1-linked ALS and potentially also in related motor neuron diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0111-6) contains supplementary material, which is available to authorized users.

Data for the effects of ER and Golgi stresses on the ER-Golgi SNARE Syntaxin5 expression and on the βAPP processing in cultured hippocampal neurons.

This paper reports the data for the effects of organelle stresses on the ER–Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER–Golgi SNAREs) syntaxin 5 (Syx5) in neuronal cells. Quantitative as well as qualitative data are presented here to verify the upregulation of Syntaxin 5 (Syx5) under ER and Golgi stresses in hippocampal neurons. Changes in the processing of β-amyloid precursor protein (βAPP) under ER stress were analyzed by immunological assays. In addition, our data shows the specific increase of Syx5 expression under ER and Golgi stresses. Interpretation of our data and further extensive insights into the role of Syx5 in βAPP processing under organelle stress can be found in "ER and Golgi stresses increase ER-Golgi SNARE Syntaxin5: Implications for organelle stress and βAPP processing" [1].

ER and Golgi stresses increase ER–Golgi SNARE Syntaxin5: Implications for organelle stress and βAPP processing

Unresolved endoplasmic reticulum (ER) stress causes neuronal death and has been implicated in neurodegenerative conditions such as Alzheimer's disease (AD). However, the mechanisms by which stress signals propagate from the ER through the Golgi apparatus and their effects on the transport and processing of AD-related proteins, such as β-amyloid precursor protein (βAPP), are unknown. We recently found that in the NG108-15 cell line, ER stress upregulates ER–Golgi-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER–Golgi SNAREs) Syx5 and Bet1. In the present study, we examined the effects of apoptosis and ER stress inducers on the expression of ER–Golgi SNARE proteins and cell viability in a primary culture of rat hippocampal neurons. An apoptosis inducer significantly downregulated the expression of ER–Golgi SNARE Syx5. ER-stress inducers upregulated the expression of Syx5 isoforms and Bet1 proteins via de novo synthesis of their mRNA transcripts. Knockdown of Syx5 during apoptosis or ER stress induction enhanced vulnerability of neurons. Additionally, we examined the effects of Golgi stress on Syx5 expression and βAPP processing. Golgi stress also induced upregulation of ER–Golgi SNARE Syx5, and concomitantly, suppressed amyloid-β peptide secretion. These findings suggest that Syx5 is a potential stress responsive factor that participates in βAPP processing and the survival pathways of neuronal cells.

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28's SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.

Btn3 regulates the endosomal sorting function of the yeast Ent3 epsin, an adaptor for SNARE proteins.

Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain-containing proteins involved in protein trafficking between the Golgi and late endosomes. They interact with clathrin, clathrin adaptors at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein-protein interaction screen. We identified Btn3 (also known as Tda3), a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten-disease-linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the late endosome to the Golgi. We show that Btn3 endosomal localization depends on the epsins Ent3 and Ent5. We demonstrated that in btn3Δ mutant cells, endosomal sorting of ubiquitylated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten-disease-linked protein Btn2.

Deficiency of the Cog8 Subunit in Normal and CDG‐Derived Cells Impairs the Assembly of the COG and Golgi SNARE Complexes

Multiple mutations in different subunits of the tethering complex Conserved Oligomeric Golgi (COG) have been identified as a cause for Congenital Disorders of Glycosylation (CDG) in humans. Yet, the mechanisms by which COG mutations induce the pleiotropic CDG defects have not been fully defined. By detailed analysis of Cog8 deficiency in either HeLa cells or CDG-derived fibroblasts, we show that Cog8 is required for the assembly of both the COG complex and the Golgi Stx5-GS28-Ykt6-GS15 and Stx6-Stx16-Vti1a-VAMP4 SNARE complexes. The assembly of these SNARE complexes is also impaired in cells derived from a Cog7-deficient CDG patient. Likewise, the integrity of the COG complex is also impaired in Cog1-, Cog4- and Cog6-depleted cells. Significantly, deficiency of Cog1, Cog4, Cog6 or Cog8 distinctly influences the production of COG subcomplexes and their Golgi targeting. These results shed light on the structural organization of the COG complex and its subcellular localization, and suggest that its integrity is required for both tethering of transport vesicles to the Golgi apparatus and the assembly of Golgi SNARE complexes. We propose that these two key functions are generally and mechanistically impaired in COG-associated CDG patients, thereby exerting severe pleiotropic defects.

A Model for the Self-Organization of Vesicular Flux and Protein Distributions in the Golgi Apparatus

The generation of two non-identical membrane compartments via exchange of vesicles is considered to require two types of vesicles specified by distinct cytosolic coats that selectively recruit cargo, and two membrane-bound SNARE pairs that specify fusion and differ in their affinities for each type of vesicles. The mammalian Golgi complex is composed of 6–8 non-identical cisternae that undergo gradual maturation and replacement yet features only two SNARE pairs. We present a model that explains how distinct composition of Golgi cisternae can be generated with two and even a single SNARE pair and one vesicle coat. A decay of active SNARE concentration in aging cisternae provides the seed for a cis trans SNARE gradient that generates the predominantly retrograde vesicle flux which further enhances the gradient. This flux in turn yields the observed inhomogeneous steady-state distribution of Golgi enzymes, which compete with each other and with the SNAREs for incorporation into transport vesicles. We show analytically that the steady state SNARE concentration decays exponentially with the cisterna number. Numerical solutions of rate equations reproduce the experimentally observed SNARE gradients, overlapping enzyme peaks in cis, medial and trans and the reported change in vesicle nature across the Golgi: Vesicles originating from younger cisternae mostly contain Golgi enzymes and SNAREs enriched in these cisternae and extensively recycle through the Endoplasmic Reticulum (ER), while the other subpopulation of vesicles contains Golgi proteins prevalent in older cisternae and hardly reaches the ER.