gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.     

Retrograde Transport from the Pre-Golgi Intermediate Compartment and the Golgi Complex Is Affected by the Vacuolar H+-ATPase Inhibitor Bafilomycin A1

The effect of the vacuolar H⁺-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments. Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected. Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive ~80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments. In parallel, redistribution of β-coatomer protein from the Golgi to peripheral pre-Golgi structures took place. The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport. These results suggest that the pre-Golgi structures contain an active H⁺-ATPase that regulates retrograde transport at the ER–Golgi boundary. Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.     

Colocalization of Ca2+-ATPase and GRP94 with p58 and the effects of thapsigargin on protein recycling suggest the participation of the pre-Golgi intermediate compartment in intracellular Ca2+ storage

We have studied the localization of functional components of cellular Ca2+ transport and storage and the effects of thapsigargin (TG), a specific inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), with respect to the p58-containing pre-Golgi intermediate compartment (IC). The depletion of Ca2+ stores in normal rat kidney (NRK) cells by TG abolished the retention of the KDEL-containing, Ca2+-binding, luminal ER chaperones GRP94/endoplasmin and GRP78/BiP, and resulted in the appearance of the proteins in the culture medium before inducing their synthesis. Immunolocalization of GRP94 in TG-treated cells showed that the protein was transported to the Golgi complex and, in parallel, the KDEL receptor was redistributed from the Golgi to p58-positive IC structures, but was not transported further to the ER. Similarly, p58 that normally cycles between the ER, IC, and cis-Golgi, was largely depleted from the cell periphery and arrested in large-sized IC elements and numerous vesicles or buds in the Golgi region, showing that TG selectively blocks its recycling from the IC back to the ER. Importantly, cell fractionation analyses and confocal fluorescence microscopy provided evidence that the IC elements in unperturbed cells contain SERCA and a considerable pool of GRP94. Thus, the observed effects of TG on protein retention and recycling can be explained by a change in the luminal Ca2+ concentration of the IC. Moreover, the compositional properties of the IC elements suggest that they participate in intracellular Ca2+ storage.     

Okadaic Acid Induces Selective Arrest of Protein Transport in the Rough Endoplasmic Reticulum and Prevents Export into COPII-Coated Structures

Quantitative immunoelectron microscopy and subcellular fractionation established the site of endoplasmic reticulum (ER)-Golgi transport arrest induced by the phosphatase inhibitor okadaic acid (OA). OA induced the disappearance of transitional element tubules and accumulation of the anterograde-transported Chandipura (CHP) virus G protein only in the rough ER (RER) and not at more distal sites. The block was specific to the early part of the anterograde pathway, because CHP virus G protein that accumulated in the intermediate compartment (IC) at 15°C could gain access to Golgi stack enzymes. OA also induced RER accumulation of the IC protein p53/p58 via an IC-RER recycling pathway which was resistant to OA and inhibited by the G protein activator aluminium fluoride. The role of COPII coats in OA transport block was investigated by using immunofluorescence and cell fractionation. In untreated cells the COPII coat protein sec 13p colocalized with p53/p58 in Golgi-IC structures of the juxtanuclear region and peripheral cytoplasm. During OA treatment, p53/p58 accumulated in the RER but was excluded from sec 13p-containing membrane structures. Taken together our data indicate that OA induces an early defect in RER export which acts to prevent entry into COPII-coated structures of the IC region.     

An InCytes from MBC Selection: The Function of the Intermediate Compartment in Pre-Golgi Trafficking Involves its Stable Connection with the Centrosome

Because the functional borders of the intermediate compartment (IC) are not well defined, the spatial map of the transport machineries operating between the endoplasmic reticulum (ER) and the Golgi apparatus remains incomplete. Our previous studies showed that the IC consists of interconnected vacuolar and tubular parts with specific roles in pre-Golgi trafficking. Here, using live cell imaging, we demonstrate that the tubules containing the GTPase Rab1A create a long-lived membrane compartment around the centrosome. Separation of this pericentrosomal domain of the IC from the Golgi ribbon, due to centrosome motility, revealed that it contains a distinct pool of COPI coats and acts as a temperature-sensitive way station in post-ER trafficking. However, unlike the Golgi, the pericentrosomal IC resists the disassembly of COPI coats by brefeldin A, maintaining its juxtaposition with the endocytic recycling compartment, and operation as the focal point of a dynamic tubular network that extends to the cell periphery. These results provide novel insight into the compartmental organization of the secretory pathway and Golgi biogenesis. Moreover, they reveal a direct functional connection between the IC and the endosomal system, which evidently contributes to unconventional transport of the cystic fibrosis transmembrane conductance regulator to the cell surface.     

Atypical protein kinase C plays a critical role in protein transport from pre-Golgi intermediates

The small GTPase Rab2 requires atypical protein kinase C iota/lambda (PKCiota/lambda) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702-712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCiota/lambda in the early secretory pathway. A peptide corresponding to the unique PKCiota/lambda pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCiota/lambda, beta-COP, and p53/p58. Because Rab2, beta-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCiota/lambda (W274K). Both the pseudosubstrate peptide and kinase-dead PKCiota/lambda in tandem with Rab2 caused sustained membrane association of PKCiota/lambda, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCiota/lambda was reversed by PKCiota/lambda wild type. These combined results indicate that PKCiota/lambda is essential for protein transport in the early secretory pathway and suggest that PKCiota/lambda kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.     

A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment.

Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers.     

Regulation of the cell surface expression and function of angiotensin II type 1 receptor by Rab1-mediated endoplasmic reticulum-to-Golgi transport in cardiac myocytes

Rab1 GTPase coordinates vesicle-mediated protein transport specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. We recently demonstrated that Rab1 is involved in the export of angiotensin II (Ang II) type 1 receptor (AT1R) to the cell surface in HEK293 cells and that transgenic mice overexpressing Rab1 in the myocardium develop cardiac hypertrophy. To expand these studies, we determined in this report whether the modification of Rab1-mediated ER-to-Golgi transport can alter the cell surface expression and function of endogenous AT1R and AT1R-mediated hypertrophic growth in primary cultures of neonatal rat ventricular myocytes. Adenovirus-mediated gene transfer of wild-type Rab1 (Rab1WT) significantly increased cell surface expression of endogenous AT1R in neonatal cardiomyocytes, whereas the dominant-negative mutant Rab1N124I had the opposite effect. Brefeldin A treatment blocked the Rab1WT-induced increase in AT1R cell surface expression. Fluorescence analysis of the subcellular localization of AT1R revealed that Rab1 regulated AT1R transport specifically from the ER to the Golgi in HL-1 cardiomyocytes. Consistent with their effects on AT1R export, Rab1WT and Rab1N124I differentially modified the AT1R-mediated activation of ERK1/2 and its upstream kinase MEK1. More importantly, adenovirus-mediated expression of Rab1N124I markedly attenuated the Ang II-stimulated hypertrophic growth as measured by protein synthesis, cell size, and sarcomeric organization in neonatal cardiomyocytes. In contrast, Rab1WT expression augmented the Ang II-mediated hypertrophic response. These data strongly indicate that AT1R function in cardiomyocytes can be modulated through manipulating AT1R traffic from the ER to the Golgi and provide the first evidence implicating the ER-to-Golgi transport as a regulatory site for control of cardiomyocyte growth.     

Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway.

The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-DeltaF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000) Traffic 1, 852-870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNAp REceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells.     

A role for the Rab6B Bicaudal-D1 interaction in retrograde transport in neuronal cells

The Rab6 subfamily of small GTPases consists of three different isoforms: Rab6A, Rab6A' and Rab6B. Both Rab6A and Rab6A' are ubiquitously expressed whereas Rab6B is predominantly expressed in brain. Recent studies have shown that Rab6A' is the isoform regulating the retrograde transport from late endosomes via the Golgi to the ER and in the transition from anaphase to metaphase during mitosis. Since the role of Rab6B is still ill defined, we set out to characterize its intracellular environment and dynamic behavior. In a Y-2H search for novel Rab6 interacting proteins, we identified Bicaudal-D1, a large coiled-coil protein known to bind to the dynein/dynactin complex and previously shown to be a binding partner for Rab6A/Rab6A'. Co-immunoprecipitation studies and pull down assays confirmed that Bicaudal-D1 also interacts with Rab6B in its active form. Using confocal laser scanning microscopy it was established that Rab6B and Bicaudal-D1 co-localize at the Golgi and vesicles that align along microtubules. Furthermore, both proteins co-localized with dynein in neurites of SK-N-SH cells. Live cell imaging revealed bi-directional movement of EGFP-Rab6B structures in SK-N-SH neurites. We conclude from our data that the brain-specific Rab6B via Bicaudal-D1 is linked to the dynein/dynactin complex, suggesting a regulatory role for Rab6B in the retrograde transport of cargo in neuronal cells.     

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.     

COPI recruitment is modulated by a Rab1b-dependent mechanism

The small GTPase Rab1b is essential for endoplasmic reticulum (ER) to Golgi transport, but its exact function remains unclear. We have examined the effects of wild-type and three mutant forms of Rab1b in vivo. We show that the inactive form of Rab1b (the N121I mutant with impaired guanine nucleotide binding) blocks forward transport of cargo and induces Golgi disruption. The phenotype is analogous to that induced by brefeldin A (BFA): it causes resident Golgi proteins to relocate to the ER and induces redistribution of ER-Golgi intermediate compartment proteins to punctate structures. The COPII exit machinery seems to be functional in cells expressing the N121I mutant, but COPI is compromised, as shown by the release of beta-COP into the cytosol. Our results suggest that Rab1b function influences COPI recruitment. In support of this, we show that the disruptive effects of N121I can be reversed by expressing known mediators of COPI recruitment, the GTPase ARF1 and its guanine nucleotide exchange factor GBF1. Further evidence is provided by the finding that cells expressing the active form of Rab1b (the Q67L mutant with impaired GTPase activity) are resistant to BFA. Our data suggest a novel role for Rab1b in ARF1- and GBF1-mediated COPI recruitment pathway.     

Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner

During membrane traffic, transport carriers are first tethered to the target membrane prior to undergoing fusion. Mechanisms exist to connect tethering with fusion, but in most cases, the details remain poorly understood. GM130 is a member of the golgin family of coiled-coil proteins tat is involved in membrane tethering at the endoplasmic reticulum (ER) to Golgi intermediate compartment and cis-Golgi. Here, we demonstrate that GM130 interacts with syntaxin 5, a t-SNARE also localized to the early secretory pathway. Binding to syntaxin 5 is specific, direct, and mediated by the membrane-proximal region of GM130. Interestingly, interaction with syntaxin 5 is inhibited by the binding of the vesicle docking protein p115 to a distal binding site in GM130. The interaction between GM130 and the small GTPase Rab1 is also inhibited by p115 binding. Our findings suggest a mechanism for coupling membrane tethering and fusion at the ER to Golgi intermediate compartment and cis-Golgi, with GM130 playing a central role in linking these processes. Consistent with this hypothesis, we find that depletion of GM130 by RNA interference slows the rate of ER to Golgi trafficking in vivo. The interactions of GM130 with syntaxin 5 and Rab1 are also regulated by mitotic phosphorylation, which is likely to contribute to the inhibition of ER to Golgi trafficking that occurs when mammalian cells enter mitosis.     

Golgi-bound Rab34 is a novel member of the secretory pathway

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.     

Cis-Golgi matrix proteins move directly to endoplasmic reticulum exit sites by association with tubules

The role of cis-medial Golgi matrix proteins in retrograde traffic is poorly understood. We have used imaging techniques to understand the relationship between the cis-medial Golgi matrix and transmembrane proteins during retrograde traffic in control and brefeldin A (BFA)-treated cells. All five of the cis-medial matrix proteins tested were associated with retrograde tubules within 2-3 min of initiation of tubule formation. Then, at later time points (3-10 min), transmembrane proteins are apparent in the same tubules. Strikingly, both the matrix proteins and the transmembrane proteins moved directly to endoplasmic reticulum (ER) exit sites labeled with p58 and Sec13, and there seemed to be a specific interaction between the ER exit sites and the tips or branch points of the tubules enriched for the matrix proteins. After the initial interaction, Golgi matrix proteins accumulated rapidly (5-10 min) at ER exit sites, and Golgi transmembrane proteins accumulated at the same sites approximately 2 h later. Our data suggest that Golgi cis-medial matrix proteins participate in Golgi-to-ER traffic and play a novel role in tubule formation and targeting.     

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.     

GTRAP3-18 serves as a negative regulator of Rab1 in protein transport and neuronal differentiation

Glutamate transporter associated protein 3–18 (GTRAP3-18) is an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family interacting with small Rab GTPases, which regulate intracellular trafficking events. Its impact on secretory trafficking has not been investigated. We report here that GTRAP3-18 has an inhibitory effect on Rab1, which is involved in ER-to-Golg trafficking. The effects on the early secretory pathway in HEK293 cells were: reduction of the rate of ER-to-Golgi transport of the vesicular stomatitis virus glycoprotein (VSVG), slowed accumulation of a Golgi marker plasmid in pre-Golgi structures after Brefeldin A treatment and inhibition of cargo concentration of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) into transpor complexes in HEK293 cells, an effect that could be completely reversed in the presence of an excess of Rab1. In accordance with the known role of Rab1 in neurite formation, overexpression of GTRAP3-18 significantly inhibited the length of outgrowing neurites in differentiated CAD cells. The inhibitory effect of GTRAP3-18 on neurite growth was rescued by co-expression with Rab1, supporting the conclusion that GTRAP 3-18 acted by inhibiting Rab1 action. Finally, we hypothesized that expression of GTRAP3-18 in the brain shoul be lower at stages of active synaptogenesis compared to early developmental stages. This was the case as expression of GTRAP3-18 declined from E17 to P0 and adult rat brains. Thus, we propose a model where protein trafficking and neuronal differentiation are directly linked by the interaction of Rab1 and its regulator GTRAP3-18.     

Rab6 regulates both ZW10/RINT-1 and conserved oligomeric Golgi complex-dependent Golgi trafficking and homeostasis

We used multiple approaches to investigate the role of Rab6 relative to Zeste White 10 (ZW10), a mitotic checkpoint protein implicated in Golgi/endoplasmic reticulum (ER) trafficking/transport, and conserved oligomeric Golgi (COG) complex, a putative tether in retrograde, intra-Golgi trafficking. ZW10 depletion resulted in a central, disconnected cluster of Golgi elements and inhibition of ERGIC53 and Golgi enzyme recycling to ER. Small interfering RNA (siRNA) against RINT-1, a protein linker between ZW10 and the ER soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 18, produced similar Golgi disruption. COG3 depletion fragmented the Golgi and produced vesicles; vesicle formation was unaffected by codepletion of ZW10 along with COG, suggesting ZW10 and COG act separately. Rab6 depletion did not significantly affect Golgi ribbon organization. Epistatic depletion of Rab6 inhibited the Golgi-disruptive effects of ZW10/RINT-1 siRNA or COG inactivation by siRNA or antibodies. Dominant-negative expression of guanosine diphosphate-Rab6 suppressed ZW10 knockdown induced-Golgi disruption. No cross-talk was observed between Rab6 and endosomal Rab5, and Rab6 depletion failed to suppress p115 (anterograde tether) knockdown-induced Golgi disruption. Dominant-negative expression of a C-terminal fragment of Bicaudal D, a linker between Rab6 and dynactin/dynein, suppressed ZW10, but not COG, knockdown-induced Golgi disruption. We conclude that Rab6 regulates distinct Golgi trafficking pathways involving two separate protein complexes: ZW10/RINT-1 and COG.     

GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus

The subcellular localization, interacting partners, and function of GS15, a Golgi SNARE, remain to be established. In our present study, it is revealed that unlike proteins (Bet1 and the KDEL receptor) cycling between the Golgi and the intermediate compartment (IC, inclusive of the ER exit sites), GS15 is not redistributed into the IC upon incubation at 15 degrees C or when cells are treated with brefeldin A. Immuno-electron microscopy (immuno-EM) reveals that GS15 is mainly found in the medial-cisternae of the Golgi apparatus and adjacent tubulo-vesicular elements. Coimmunoprecipitation experiments suggest that GS15 exists in a distinct SNARE complex that contains SNAREs (syntaxin5, GS28, and Ykt6) that are implicated in both ER-to-Golgi and intra-Golgi transport but not with SNAREs involved exclusively in ER-to-Golgi traffic. Furthermore, components of COPI coat can be selectively coimmunoprecipitated with GS15 from Golgi extracts. Overexpression of mutant forms of GS15 affects the normal distribution of cis- and medial-Golgi proteins (GS28, syntaxin 5, and Golgi mannosidase II), whereas proteins of the trans-Golgi and TGN (Vti1-rp2/Vti1a and syntaxin 6) and Golgi matrix/scaffold (GM130 and p115) are less affected. When the level of GS15 is reduced by duplex 21-nt small interfering RNA (siRNA)-mediated knockdown approach, diverse markers of the Golgi apparatus are redistributed into small dotty and diffuse labeling, suggesting an essential role of GS15 in the Golgi apparatus.