Rab33b and Rab6 are Functionally Overlapping Regulators of Golgi Homeostasis and Trafficking

We used multiple approaches to investigate the coordination of trans and medial Rab proteins in the regulation of intra‐Golgi retrograde trafficking. We reasoned that medially located Rab33b might act downstream of the trans Golgi Rab, Rab6, in regulating intra‐Golgi retrograde trafficking. We found that knockdown of Rab33b, like Rab6, suppressed conserved oligomeric Golgi (COG) complex‐ or Zeste White 10 (ZW10)‐depletion induced disruption of the Golgi ribbon in HeLa cells. Moreover, efficient GTP‐restricted Rab6 induced relocation of Golgi enzymes to the endoplasmic reticulum (ER) was Rab33b‐dependent, but not vice versa, suggesting that the two Rabs act sequentially in an intra‐Golgi Rab cascade. In support of this hypothesis, we found that overexpression of GTP‐Rab33b induced the dissociation of Rab6 from Golgi membranes in vivo. In addition, the transport of Shiga‐like toxin B fragment (SLTB) from the trans to cis Golgi and ER required Rab33b. Surprisingly, depletion of Rab33b had little, if any, immediate effect on cell growth and multiplication. Furthermore, anterograde trafficking of tsO45G protein through the Golgi apparatus was normal. We suggest that the Rab33b/Rab6 regulated intra‐Golgi retrograde trafficking pathway must coexist with other Golgi trafficking pathways. In conclusion, we provide the first evidence that Rab33b and Rab6 act to coordinate a major intra‐Golgi retrograde trafficking pathway. This coordination may have parallels with Rab conversion/cascade events that regulate endosome, phagosome and exocytic processes.     

Distinct Sets of Rab6 Effectors Contribute to ZW10‐ and COG‐Dependent Golgi Homeostasis

The organization of the Golgi apparatus is determined in part by the interaction of Rab proteins and their diverse array of effectors. Here, we used multiple approaches to identify and characterize a small subset of effectors that mimicked the effects of Rab6 on Golgi ribbon organization. In a visual‐based, candidate protein screen, we found that the individual depletion of any of three Rab6 effectors, myosin IIA (MyoIIA), Kif20A and Bicaudal D (BicD), was sufficient to suppress Golgi ribbon fragmentation/dispersal coupled to retrograde tether proteins in a manner paralleling Rab6. MyoIIA and Kif20A depletions were pathway selective and suppressed ZW10‐dependent Golgi ribbon fragmentation/dispersal only whereas BicD depletion, like Rab6, suppressed both ZW10‐ and COG‐dependent Golgi ribbon fragmentation. The MyoIIA effects could be produced in short‐term assays by the reversible myosin inhibitor, blebbistatin. At the electron microscope level, the effects of BicD‐depletion mimicked many of those of Rab6‐depletion: longer and more continuous Golgi cisternae and a pronounced accumulation of coated vesicles. Functionally, BicD‐depleted cells were inhibited in transport of newly synthesized VSV‐G protein to the cell surface. In summary, our results indicate small, partially overlapping subsets of Rab6 effectors are differentially important to two tether‐dependent pathways essential to Golgi organization and function.      

Electron tomography reveals Rab6 is essential to the trafficking of trans-Golgi clathrin and COPI-coated vesicles and the maintenance of Golgi cisternal number

We have shown previously that Rab6, a small, trans-Golgi-localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to coat protein I (COPI)-coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans-Golgi/trans-Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6-depleted HeLa cells that we observed may well be coupled to the trans-Golgi accumulation of COPI-coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6-dependent protein machine affecting Golgi-proximal, coated vesicle accumulation and probably transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.     

RINT-1 regulates the localization and entry of ZW10 to the syntaxin 18 complex

RINT-1 was first identified as a Rad50-interacting protein that participates in radiation-induced G2/M checkpoint control. We have recently reported that RINT-1, together with the dynamitin-interacting protein ZW10 and others, is associated with syntaxin 18, an endoplasmic reticulum (ER)-localized SNARE involved in membrane trafficking between the ER and Golgi. To address the role of RINT-1 in membrane trafficking, we examined the effects of overexpression and knockdown of RINT-1 on Golgi morphology and protein transport from the ER. Overexpression of the N-terminal region of RINT-1, which is responsible for the interaction with ZW10, caused redistribution of ZW10. Concomitantly, ER-to-Golgi transport was blocked and the Golgi was dispersed. Knockdown of RINT-1 also disrupted membrane trafficking between the ER and Golgi. Notably, silencing of RINT-1 resulted in a reduction in the amount of ZW10 associated with syntaxin 18, concomitant with ZW10 redistribution. In contrast, no redistribution or release of RINT-1 from the syntaxin 18 complex was observed when ZW10 expression was reduced. These results taken together suggest that RINT-1 coordinates the localization and function of ZW10 by serving as a link between ZW10 and the SNARE complex comprising syntaxin 18.     

The COG Complex, Rab6 and COPI Define a Novel Golgi Retrograde Trafficking Pathway that is Exploited by SubAB Toxin

Toxin trafficking studies provide valuable information about endogenous pathways of intracellular transport. Subtilase cytotoxin (SubAB) is transported in a retrograde manner through the endosome to the Golgi and then to the endoplasmic reticulum (ER), where it specifically cleaves the ER chaperone BiP/GRP78 (Binding immunoglobin protein/Glucose-Regulated Protein of 78 kDa). To identify the SubAB Golgi trafficking route, we have used siRNA-mediated silencing and immunofluorescence microscopy in HeLa and Vero cells. Knockdown (KD) of subunits of the conserved oligomeric Golgi (COG) complex significantly delays SubAB cytotoxicity and blocks SubAB trafficking to the cis Golgi. Depletion of Rab6 and β-COP proteins causes a similar delay in SubAB-mediated GRP78 cleavage and did not augment the trafficking block observed in COG KD cells, indicating that all three Golgi factors operate on the same 'fast' retrograde trafficking pathway. SubAB trafficking is completely blocked in cells deficient in the Golgi SNARE Syntaxin 5 and does not require the activity of endosomal sorting nexins SNX1 and SNX2. Surprisingly, depletion of Golgi tethers p115 and golgin-84 that regulates two previously described coat protein I (COPI) vesicle-mediated pathways did not interfere with SubAB trafficking, indicating that SubAB is exploiting a novel COG/Rab6/COPI-dependent retrograde trafficking pathway.     

Implication of ZW10 in membrane trafficking between the endoplasmic reticulum and Golgi

ZW10, a dynamitin-interacting protein associated with kinetochores, is known to participate directly in turning off of the spindle checkpoint. In the present study, we show that ZW10 is located in the endoplasmic reticulum as well as in the cytosol during interphase, and forms a subcomplex with RINT-1 (Rad50-interacting protein) and p31 in a large complex comprising syntaxin 18, an endoplasmic reticulum-localized t-SNARE implicated in membrane trafficking. Like conventional syntaxin-binding proteins, ZW10, RINT-1 and p31 dissociated from syntaxin 18 upon Mg(2+)-ATP treatment in the presence of NSF and alpha-SNAP, whereas the subcomplex was not disassembled. Overexpression, microinjection and knockdown experiments revealed that ZW10 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. The present results disclose an unexpected role for a spindle checkpoint protein, ZW10, during interphase.     

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.     

Correlation of Golgi localization of ZW10 and centrosomal accumulation of dynactin

ZW10 participates in the termination of the spindle checkpoint during mitosis by interacting with dynamitin, a subunit of the dynein accessory complex dynactin. We previously showed that ZW10 is attached to the endoplasmic reticulum through RINT-1 in interphase HeLa cells and involved in membrane transport between the endoplasmic reticulum and Golgi. Although a recent study demonstrated that ZW10 is localized in the Golgi in COS7 cells, the mechanism that regulates ZW10 localization remains unknown. In this study we showed a correlation between the Golgi localization of ZW10 and the centrosomal accumulation of dynactin. The amounts of ZW10 associated with dynactin were larger in cells where ZW10 was present in the Golgi than those where ZW10 was not in the Golgi. The targeting of ZW10 to the perinuclear Golgi region was found to depend on the perinuclear accumulation of dynactin, suggesting that dynactin regulates ZW10 localization.     

Rab41 Is a Novel Regulator of Golgi Apparatus Organization That Is Needed for ER-To-Golgi Trafficking and Cell Growth

Background

The 60⁺ members of the mammalian Rab protein family group into subfamilies postulated to share common functionality. The Rab VI subfamily contains 5 Rab proteins, Rab6a/a’, Rab6b, Rab6c and Rab41. High-level knockdown of Rab6a/a’ has little effect on the tightly organized Golgi ribbon in HeLa cells as seen by fluorescence microscopy. In striking contrast, we found Rab41 was strongly required for normal Golgi ribbon organization.

Methods/Results

Treatment of HeLa cells with Rab41 siRNAs scattered the Golgi ribbon into clustered, punctate Golgi elements. Overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a similar Golgi phenotype. By electron microscopy, Rab41 depletion produced short, isolated Golgi stacks. Golgi-associated vesicles accumulated. At low expression levels, wild type and GTP-locked Rab41 showed little concentration in the Golgi region, but puncta were observed and most were in ruffled regions at the cell periphery. There was 25% co-localization of GTP-locked Rab41 with the ER marker, Sec61p. GDP-locked Rab41, as expected, displayed an entirely diffuse cytoplasmic distribution. Depletion of Rab41 or overexpression of GDP-locked Rab41 partially inhibited ER-to-Golgi transport of VSV-G protein. However, Rab41 knockdown had little, if any, effect on endosome-to-Golgi transport of SLTB. Additionally, after a 2-day delay, treatment with Rab41 siRNA inhibited cell growth, while overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a rapid, progressive cell loss. In double knockdown experiments with Rab6, the Golgi ribbon was fragmented, a result consistent with Rab41 and Rab6 acting in parallel.

Conclusion

We provide the first evidence for distinctive Rab41 effects on Golgi organization, ER-to-Golgi trafficking and cell growth. When combined with the evidence that Rab6a/a’ and Rab6b have diverse roles in Golgi function, while Rab6c regulates mitotic function, our data indicate that Rab VI subfamily members, although related by homology and structure, share limited functional conservation.     

Acute COG complex inactivation unveiled its immediate impact on Golgi and illuminated the nature of intra-Golgi recycling vesicles

Conserved Oligomeric Golgi (COG) complex controls Golgi trafficking and glycosylation, but the precise COG mechanism is unknown. The auxin-inducible acute degradation system was employed to investigate initial defects resulting from COG dysfunction. We found that acute COG inactivation caused a massive accumulation of COG-dependent (CCD) vesicles that carry the bulk of Golgi enzymes and resident proteins. v-SNAREs (GS15, GS28) and v-tethers (giantin, golgin84, and TMF1) were relocalized into CCD vesicles, while t-SNAREs (STX5, YKT6), t-tethers (GM130, p115), and most of Rab proteins remained Golgi-associated. Airyscan microscopy and velocity gradient analysis revealed that different Golgi residents are segregated into different populations of CCD vesicles. Acute COG depletion significantly affected three Golgi-based vesicular coats—COPI, AP1, and GGA, suggesting that COG uniquely orchestrates tethering of multiple types of intra-Golgi CCD vesicles produced by different coat machineries. This study provided the first detailed view of primary cellular defects associated with COG dysfunction in human cells.     

COG-7-deficient Human Fibroblasts Exhibit Altered Recycling of Golgi Proteins

Recently, we reported that two siblings presenting with the clinical syndrome congenital disorders of glycosylation (CDG) have mutations in the gene encoding Cog7p, a member of the conserved oligomeric Golgi (COG) complex. In this study, we analyzed the localization and trafficking of multiple Golgi proteins in patient fibroblasts under a variety of conditions. Although the immunofluorescent staining pattern of several Golgi proteins was indistinguishable from normal, the staining of endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC)-53 and the vesicular-soluble N-ethylmaleimide-sensitive factor attachment protein receptors GS15 and GS28 was abnormal, and the steady-state level of GS15 was greatly decreased. Retrograde transport of multiple Golgi proteins to the ER in patient fibroblasts via brefeldin A-induced tubules was significantly slower than occurs in normal fibroblasts, whereas anterograde protein trafficking was much less affected. After prolonged treatment with brefeldin A, several Golgi proteins were detected in clusters that colocalize with the microtubule-organizing center in patient cells. All of these abnormalities were normalized in COG7-corrected patient fibroblasts. These results serve to better define the role of the COG complex in facilitating protein trafficking between the Golgi and ER and provide a diagnostic framework for the identification of CDG defects involving trafficking proteins.     

Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.     

Distinct pathways for the trafficking of angiotensin II and adrenergic receptors from the endoplasmic reticulum to the cell surface: Rab1-independent transport of a G protein-coupled receptor

The molecular mechanism underlying the transport of G protein-coupled receptors from the endoplasmic reticulum (ER) to the cell surface is poorly understood. This issue was addressed by determining the role of Rab1, a Ras-related small GTPase that coordinates vesicular protein transport in the early secretory pathway, in the subcellular distribution and function of the angiotensin II type 1A receptor (AT1R), beta2-adrenergic receptor (AR), and alpha2B-AR in HEK293T cells. Inhibition of endogenous Rab1 function by transient expression of dominant-negative Rab1 mutants or Rab1 small interfering RNA (siRNA) induced a marked perinuclear accumulation and a significant reduction in cell-surface expression of AT1R and beta2-AR. The accumulated receptors were colocalized with calregulin (an ER marker) and GM130 (a Golgi marker), consistent with Rab1 function in regulating protein transport from the ER to the Golgi. In contrast, dominant-negative Rab1 mutants and siRNA had no effect on the subcellular distribution of alpha2B-AR. Similarly, expression of dominant-negative Rab1 mutants and siRNA depletion of Rab1 significantly attenuated AT1R-mediated inositol phosphate accumulation and ERK1/2 activation and beta2-AR-mediated ERK1/2 activation, but not alpha2B-AR-stimulated ERK1/2 activation. These data indicate that Rab1 GTPase selectively regulates intracellular trafficking and signaling of G protein-coupled receptors and suggest a novel, as yet undefined pathway for movement of G protein-coupled receptors from the ER to the cell surface.     

A positive signal prevents secretory membrane cargo from recycling between the Golgi and the ER

The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward‐bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi‐to‐plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal‐deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tail‐anchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy‐ and Rab6‐dependent, and Rab6 inhibition accelerated signal‐deleted VSVG's transport to the cell surface. Our results extend the dynamic bi‐directional relationship between the Golgi and ER to include surface‐directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.     

Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of {beta}1,4-galactosyltransferase 1 and {alpha}2,6-sialyltransferase 1

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure.     

The conserved oligomeric Golgi complex is required for fucosylation of N-glycans in Caenorhabditis elegans

The conserved oligomeric Golgi complex (COG) is a hetero-octomeric peripheral membrane protein required for retrograde vesicular transport and glycoconjugate biosynthesis within the Golgi. Mutations in subunits 1, 4, 5, 6, 7 and 8 are the basis for a rare inheritable human disease termed congenital disorders of glycosylation type-II. Defects to COG complex function result in aberrant glycosylation, protein trafficking and Golgi structure. The cellular function of the COG complex and its role in protein glycosylation are not completely understood. In this study, we report the first detailed structural analysis of N-glycans from a COG complex-deficient organism. We employed sequential ion trap mass spectrometry of permethylated N-glycans to demonstrate that the COG complex is essential for the formation of fucose-rich N-glycans, specifically antennae fucosylated structures in Caenorhabditis elegans. Our results support the supposition that disruption to the COG complex interferes with normal protein glycosylation in the medial and/or trans-Golgi.     

Legionella hijacks the host Golgi-to-ER retrograde pathway for the association of Legionella-containing vacuole with the ER

Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6A, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6A to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6A cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins.     

Progress in studies of ZW10, a proper chromosome segregation protein

The conserved protein ZW10 is found in various organisms. It is localized on the kinetochores or spindle microtubules during cell division. ZW10 regulates not only the segregation of homologous chromosomes, each consisting of attached sister chromatids (during the first meiotic division), but also the separation of individual chromatids (during mitosis and the second meiotic division). ZW10 is required for proper chromosome segregation during both mitosis and meiosis. The effects of zwl0 mutations are similar for both equational and reductional divisions, giving rise to anaphases with lagging chromosomes and/or unequal numbers of chromosomes at the two poles. The localization of ZW10 is similar during mitosis, meiosis I, and meiosis II. In interphase the distribution of ZW10 changes; it is localized in the endoplasmic reticulum, Golgi apparatus, and in the cytosol and is involved in membrane trafficking between the endoplasmic reticulum and Golgi apparatus. ZW10 forms a subcomplex with RINT-1 and p31 which are involved in a larger complex comprising syntaxin 18, an endoplasmic reticulum-localized t-SNARE that is implicated in membrane trafficking. The text was submitted by the authors in English.     

Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5a/Sed5 enhances intra-Golgi SNARE complex stability

Tethering factors mediate initial interaction of transport vesicles with target membranes. Soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) enable consequent docking and membrane fusion. We demonstrate that the vesicle tether conserved oligomeric Golgi (COG) complex colocalizes and coimmunoprecipitates with intra-Golgi SNARE molecules. In yeast cells, the COG complex preferentially interacts with the SNARE complexes containing yeast Golgi target (t)-SNARE Sed5p. In mammalian cells, hCog4p and hCog6p interact with Syntaxin5a, the mammalian homologue of Sed5p. Moreover, fluorescence resonance energy transfer reveals an in vivo interaction between Syntaxin5a and the COG complex. Knockdown of the mammalian COG complex decreases Golgi SNARE mobility, produces an accumulation of free Syntaxin5, and decreases the steady-state levels of the intra-Golgi SNARE complex. Finally, overexpression of the hCog4p N-terminal Syntaxin5a-binding domain destabilizes intra-Golgi SNARE complexes, disrupting the Golgi. These data suggest that the COG complex orchestrates vesicular trafficking similarly in yeast and mammalian cells by binding to the t-SNARE Syntaxin5a/Sed5p and enhancing the stability of intra-Golgi SNARE complexes.     

Identification of the Neuroblastoma-amplified Gene Product as a Component of the Syntaxin 18 Complex Implicated in Golgi-to-Endoplasmic Reticulum Retrograde Transport

Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.