The Putative “Switch 2” Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the α2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative α2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the α3 and α4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B α2 helix mutants were not efficiently prenylated as determined by incorporation of [³H]mevalonate. Metabolic labeling studies using [³²P]orthophosphate indicated that the poor prenylation of the Rab1B α2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the α2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.     

Structure of Rab escort protein-1 in complex with Rab geranylgeranyltransferase

Posttranslational geranylgeranylation of Rab GTPases is catalyzed by Rab geranylgeranyltransferase (RabGGTase), which consists of a catalytic alpha/beta heterodimer and an accessory Rab escort protein (REP). The crystal structure of isoprenoid-bound RabGGTase complexed to REP-1 has been solved to 2.7 A resolution. The complex interface buries a surprisingly small surface area of ca. 680 A and is unexpectedly formed by helices 8, 10, and 12 of the RabGGTase alpha subunit and helices D and E of REP-1. We demonstrate that the affinity of RabGGTase for REP-1 is allosterically regulated by phosphoisoprenoid via a long-range trans-domain signal transduction event. Comparing the structure of REP-1 with the closely related RabGDI, we conclude that the specificity of the REP:RabGGTase interaction is defined by differently positioned phenylalanine residues conserved in the REP and GDI subfamilies.     

Cytosolic RAB3D is associated with RAB escort protein (REP), not RAB-GDP dissociation inhibitor (GDI), in dispersed chief cells from guinea pig stomach

Rab3D, a low-molecular-weight GTP-binding protein believed to be involved with regulated exocytosis, is associated with secretory granules in gastric chief cells. Although Rab3D is predominantly membrane associated, a significant fraction is cytosolic. Rab proteins are geranylgeranylated on their C-terminal cysteine motifs by geranylgeranyltransferase (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase and may accompany newly modified Rab proteins to their target membrane. In most tissues, cytosolic Rab proteins are complexed with rab-GDP dissociation inhibitor (rab-GDI). In the present study, we examined the interactions of Rab3D with cytosolic proteins in dispersed chief cells. Two REP isoforms and at least two GDI isoforms are present in chief cell and brain cytosol. When chief cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted with REP at >150 kDa, whereas rab-GDI eluted as a separate 65-kDa peak, suggesting that Rab3D exists as a complex with REP, but not with rab-GDI. In addition, a small fraction of Rab3D eluted as a monomer at 29 kDa. As has been demonstrated previously, in brain cytosol, Rab3 proteins co-elute with rab-GDI at approx. 90 kDa, suggesting that Rab3 proteins undergo active cycling between membrane and cytosolic compartments in this tissue. In vitro experiments revealed that Rab3D remains associated with REP after geranylgeranylation. Our findings suggest that, in gastric chief cells, Rab3D remains associated with REP after geranylgeranylation until it is presented to its target membrane.     

A specific feature of the angiosperm Rab escort protein (REP) and evolution of the REP/GDI superfamily

Rab GTPases participating in the regulation of vesicle trafficking in eukaryotes are geranylgeranylated by the Rab geranylgeranyl transferase (RabGGTase) in complex with the Rab escort protein (REP). Here, we describe basic properties of the Arabidopsis thaliana REP (AthREP), first REP outside yeasts or metazoans to be characterized. GFP-tagged AthREP, as well as the geranylgeranylation activity, were localized predominantly to the cytoplasm. Recombinant AthREP interacted with yeast 6His-Ypt1, tobacco 6His-RabA1a, and Arabidopsis RabA2a in vitro preferring the GDP-bound form of the latter. Recombinant AthREP with C-terminal but not N-terminal tags stimulated geranylgeranylation of various Rab GTPases in Arabidopsis extracts in vitro. Neither recombinant AthREP protein exhibited activity in yeast extracts, while recombinant yeast REP (6His-SceMrs6) stimulated Rab geranylgeranylation in all extracts tested. We found that a conserved arginine residue, R195, known to be crucial for yeast REP function, is substituted by an asparagine or threonine residue in angiosperm REPs. A point mutant allele of AthREP with arginine at this position complemented the yeast REP mutation, while wild-type AthREP did not. Based on phylogenetic analysis of REP and GDP dissociation inhibitor (GDI) sequences from a broad range of eukaryotic lineages, we propose a new view on evolution of the REP/GDI superfamily with a bi-functional REP/GDI protein as a direct ancestor.     

Molecular evolution of the Rab-escort-protein/guanine-nucleotide-dissociation-inhibitor superfamily

Prenylation of Rab GTPases regulating vesicle traffic by Rab geranylgeranyltransferase (RabGGTase) requires a complex formed by the association of newly synthesized Rab proteins with Rab-escort-protein (REP), the choroideremia-gene-product that is mutated in disease, leading to loss of vision. After delivery to the membrane by the REP-Rab complex, subsequent recycling to the cytosol requires the REP-related guanine-nucleotide-dissociation-inhibitor (GDI). Although REP and GDI share common Rab-binding properties, GDI cannot assist in Rab prenylation and REP cannot retrieve Rab proteins from the membranes. We have now isolated REP mutant proteins that are able to partially function as both REP and GDI. These results provide molecular insight into the functional and evolutionary organization of the REP/GDI superfamily.     

Thematic review series: lipid posttranslational modifications. geranylgeranylation of Rab GTPases

Rab GTPases require special machinery for protein prenylation, which include Rab escort protein (REP) and Rab geranylgeranyl transferase (RGGT). The current model of Rab geranylgeranylation proposes that REP binds Rab and presents it to RGGT. After geranylgeranylation of Rab C-terminal cysteines, REP delivers the prenylated protein to membranes. The REP-like protein Rab GDP dissociation inhibitor (RabGDI) then recycles the prenylated Rab between the membrane and the cytosol. The recent solution of crystal structures of the Rab prenylation machinery has helped to refine this model and provided further insights. The hydrophobic prenyl binding pocket of RGGT and geranylgeranyl transferase type-I (GGT-I) differs from that of farnesyl transferase (FT). A bulky tryptophan residue in FT restricts the size of the pocket, whereas in RGGT and GGT-I, this position is occupied by smaller residues. A highly conserved phenylalanine in REP, which is absent in RabGDI, is critical for the formation of the REP:RGGT complex. Finally, a geranylgeranyl binding site conserved in REP and RabGDI has been identified within helical domain II. The postprenylation events, including the specific targeting of Rabs to target membranes and the requirement for single versus double geranylgeranylation by different Rabs, remain obscure and should be the subject of future studies.     

Mouse prenylated Rab acceptor is a novel Golgi membrane protein

We have cloned a mouse prenylated Rab acceptor (mPRA), which interacts with various Rab proteins in the yeast two-hybrid system. This study investigated its intracellular localization and characterized the localization signal. The mPRA was found to be an integral membrane protein that was localized to the Golgi complex at steady state as determined by confocal fluorescence microscopy. With green fluorescent protein attached to the N-terminus of mPRA, the fusion protein was expressed in BHK cells and was shown to exhibit the same Golgi localization as the native mPRA. Systematic truncations from the N- and C-termini of mPRA revealed that the entire N-terminal half (91 residues) of the protein was dispensable for the Golgi localization. In contrast, deletion of only 5 residues from the C-terminus diminished the Golgi localization of mPRA, leading to its accumulation in the ER. The data indicate that the C-terminal half (94 residues) of mPRA is necessary and sufficient for proper folding, ER export, and Golgi localization. The Golgi localization of mPRA suggests that it may play a role in the structural organization and function of the Golgi complex.     

PRA isoforms are targeted to distinct membrane compartments

The prenylated Rab acceptor (PRA) 1 is a protein that binds prenylated Rab GTPases and inhibits their removal from the membrane by GDI. We describe here the isolation of a second isoform that can also bind Rab GTPases in a guanine nucleotide-independent manner. The two PRA isoforms showed distinct intracellular localization with PRA1 localized primarily to the Golgi complex and PRA2 to the endoplasmic reticulum (ER) compartment. The localization signal was mapped to the COOH-terminal domain of the two proteins. A DXEE motif served to target PRA1 to the Golgi. Mutation of any one of the acidic residues within this motif resulted in significant retention of PRA1 in the ER compartment. Moreover, the introduction of a di-acidic motif to the COOH-terminal domain of PRA2 resulted in partial localization to the Golgi complex. The domain responsible for ER localization of PRA2 was also confined to the carboxyl terminus. Our results showed that these sorting signals were primarily responsible for the differential localization of the two PRA isoforms.     

Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease

Choroideremia is an X-chromosome-linked disease that leads to the degeneration of the choriocapillaris, the retinal pigment epithelium and the photoreceptor layer in the eye. The gene product defective in choroideremia, CHM, is identical to Rab escort protein 1 (REP1). CHM/REP1 is an essential component of the catalytic geranylgeranyltransferase II complex (GGTrII) that delivers newly synthesized small GTPases belonging to the RAB gene family to the catalytic complex for post-translational modification. CHM/REP family members are evolutionarily related to members of the guanine nucleotide dissociation inhibitor (GDI) family, proteins involved in the recycling of Rab proteins required for vesicular membrane trafficking through the exocytic and endocytic pathways, forming the GDI/CHM superfamily. Biochemical and structural analyses have now revealed a striking parallel in the organization and function of these two families allowing us to generate a general model for GDI/CHM superfamily function in health and disease.     

Membrane topography and topogenesis of prenylated Rab acceptor (PRA1)

The mouse prenylated Rab acceptor (mPRA1) is associated with the Golgi membrane at steady state and interacts with Rab proteins. It contains two internal hydrophobic domains (34 residues each) that have enough residues to form four transmembrane (TM) segments. In this study, we have determined the membrane topography of mPRA1 in both intact cells and isolated microsomes. The putative TM segments of mPRA1 were used to substitute for a known TM segment of a model membrane protein to determine whether the mPRA1 segments integrate into the membrane. Furthermore, N-linked glycosylation scanning methods were used to distinguish luminal domains from cytoplasmic domains of mPRA1. The data demonstrate that mPRA1 is a polytopic membrane protein containing four TM segments. These TM segments act cooperatively during the translocation and integration at the endoplasmic reticulum membrane. All hydrophilic domains are in the cytoplasm, including the N-terminal domain, the linker domain between the two hydrophobic domains, and the C-terminal domain. As a result, the bulk of mPRA1 is located in the cytoplasm, supporting its postulated role in regulating Rab membrane targeting and intracellular trafficking.     

Double prenylation by RabGGTase can proceed without dissociation of the mono-prenylated intermediate

Rab geranylgeranyltransferase (RabGGTase) catalyzes the prenylation of Rab proteins. Despite possessing a single active site, RabGGTase is able to add geranylgeranyl moieties onto each of the two C-terminal cysteine residues of Rab. We have studied the kinetics of Rab double prenylation employing a combination of a novel high pressure liquid chromatography (HPLC)-based in vitro prenylation assay and fluorescence spectroscopy. Transfer of the first geranylgeranyl group proceeds with a k(1) = 0.16 s(-1), while the conversion from singly to double prenylated Rab is 4-fold slower (k(2) = 0.039 s(-1)). We found that following the first transfer reaction, the conjugated lipid is removed from the active site of RabGGTase but mono-prenylated Rab.REP complex remains bound to RabGGTase with a K(d) < 1 nm. In contrast to the doubly prenylated Rab7.REP dissociation of the mono-prenylated species from RabGGTase was only weakly stimulated by phosphoisoprenoid. Based on the obtained rate constants we calculated that at least 72% of mono-prenylated Rab molecules proceed to double prenylation without dissociating from RabGGTase. The obtained data provides an explanation of how RabGGTase discriminates between mono-prenylated intermediate and double prenylated reaction product. It also indicates that the phosphoisoprenoid acts both as a substrate and as a sensor governing the kinetics of protein.protein interactions in the double prenylation reaction.     

Expression and localization of rab escort protein isoforms in parotid acinar cells from rat

Rab proteins are geranylgeranylated on their carboxyl terminal cysteine motifs by geranylgeranyltransferase II (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase. REP may remain bound to newly isoprenylated Rab proteins and present them to their target membrane. Other studies have shown that Rab proteins cycle between the membrane and cytosolic compartments and that cytosolic Rab proteins are complexed with rab-GDI. In the present study, we examined the expression and localization of REP isoforms in parotid acinar cells. Although both REP isoforms, REP-1 and REP-2, were detected in parotid cytosol, REP-2 was the predominant isoform. Subcellular fractionation revealed that approximately 42% of cellular REP-2 is membrane-associated. REP-2 was partially removed from parotid membranes with 1 M NaCl or Na(2)CO(3), indicating that REP-2 is a peripheral membrane protein. Membrane-associated REP-2 did not colocalize with Rab3D on secretory granule membranes. However, density gradient centrifugation revealed that membrane-associated REP-2 and Rab3D colocalize on low- and high-density membrane fractions in parotid acinar cells. Isoproterenol, an agent which induces amylase release from parotid glands, caused a shift in both REP-2 and Rab3D to less dense membrane fractions. When acinar cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted exclusively with REP, not rab-GDI. In contrast, Rab1B and Rab5 eluted with both REP and Rab-GDI. Colocalization of Rab3D and REP-2 on acinar cell membranes suggests that REP-2 plays a role in delivering Rab3D to parotid membranes and may regulate guanine nucleotide binding to membrane-associated Rab3D. In addition, unlike other Rab proteins, cytosolic Rab3D appears to associate exclusively with REP, not rab-GDI in parotid acinar cells.     

The Hsp90 chaperone complex regulates GDI-dependent Rab recycling

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.     

Crystal structure of Rab geranylgeranyltransferase at 2.0 A resolution

BACKGROUND: Rab geranylgeranyltransferase (RabGGT) catalyzes the addition of two geranylgeranyl groups to the C-terminal cysteine residues of Rab proteins, which is crucial for membrane association and function of these proteins in intracellular vesicular trafficking. Unlike protein farnesyltransferase (FT) and type I geranylgeranyltransferase, which both prenylate monomeric small G proteins or short peptides, RabGGT can prenylate Rab only when Rab is in a complex with Rab escort protein (REP). RESULTS: The crystal structure of rat RabGGT at 2.0 A resolution reveals an assembly of four distinct structural modules. The beta subunit forms an alpha-alpha barrel that contains most of the residues in the active site. The alpha subunit consists of a helical domain, an immunoglobulin (Ig)-like domain, and a leucine-rich repeat (LRR) domain. The N-terminal region of the alpha subunit binds to the active site in the beta subunit; residue His2alpha directly coordinates a zinc ion. The prenyl-binding pocket of RabGGT is deeper than that in FT. CONCLUSIONS: LRR and Ig domains are often involved in protein-protein interactions; in RabGGT they might participate in the recognition and binding of REP. The binding of the N-terminal peptide of the alpha subunit to the active site suggests an autoinhibition mechanism that might contribute to the inability of RabGGT to recognize short peptides or Rab alone as its substrate. Replacement of residues Trp102beta and Tyr154beta in FT by Ser48beta and Leu99beta, respectively, in RabGGT largely determine the different lipid-binding specificities of the two enzymes.     

Crystallization and preliminary X-ray diffraction analysis of monoprenylated Rab7 GTPase in complex with Rab escort protein 1

Posttranslational geranylgeranylation of Rab GTPases is catalyzed by Rab geranylgeranyltransferase (RabGGTase), which consists of a catalytic alpha/beta heterodimer and an accessory Rab escort protein (REP). REP functions as a molecular chaperone that presents Rab proteins to the RabGGTase and after prenylation delivers them to their target membrane. Mutations in the REP-1 gene in humans lead to an X-chromosome-linked defect known as choroideremia, a progressive disease that inevitably culminates in complete blindness. Here we report in vitro assembly, purification, and crystallization of the monoprenylated Rab7GDP:REP-1 complex. X-Ray diffraction data for the REP-1:Rab7 complex were collected to 2.2-A resolution at the ESRF. The crystals belong to the orthorhombic space group P2(1)2(1)2 with unit-cell parameters a=64.3A, b=105.3A, c=132.6A. Preliminary structural analysis revealed the presence of one complex in the asymmetric unit. To understand the conformational changes in Rab protein on complex formation we also crystallized the GDP-bound form of Rab7 that diffracted to at least 1.8A on the in-house X-ray source.     

Structure of doubly prenylated Ypt1:GDI complex and the mechanism of GDI-mediated Rab recycling

In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.     

Multiple factors contribute to inefficient prenylation of Rab27a in Rab prenylation diseases

Post-translational geranylgeranylation of Rab GT-Pases is essential for their membrane association and function as regulators of intracellular vesicular transport. The reaction is catalyzed by Rab geranylgeranyltransferase (RGGT) and is assisted by the Rab escort proteins (REP), which form stable complexes with newly synthesized GDP-bound Rabs. Two genetic diseases involve the Rab geranylgeranylation machinery: choroideremia, an X-linked retinal degeneration resulting from loss-of-function mutations in REP1, and gunmetal, a mouse model of Hermansky-Pudlak syndrome resulting from mutations in the alpha-subunit of RGGT. A small subset of Rab proteins is selectively under-prenylated in both diseases, most notably Rab27a. Here we analyze why Rab27a is selectively affected in diseases of Rab geranylgeranylation. Semi-quantitative immunoblotting suggests that mass action, i.e. the amount of Rab27a relative to other Rabs, is unlikely to be a factor as the expression level of Rab27a is similar to other Rabs not affected in these diseases. In vitro binding assays and fluorescence resonance energy transfer detected by fluorescence lifetime imaging microscopy in intact cells demonstrate that Rab27a binds equally well to both REP1 and REP2, suggesting differential affinity of Rab27a for REP isoforms is not an important factor. However, steady-state kinetic analysis of the geranylgeranylation reaction indicates that REP2-Rab27a has lower affinity for RGGT compared with REP1-Rab27a. Furthermore, we show that Rab27a has relatively low GTPase activity, presumably decreasing the affinity of the REP interaction in vivo. We suggest that the restricted phenotypes observed in these diseases result from multiple contributing factors.     

Rab geranylgeranylation occurs preferentially via the pre-formed REP-RGGT complex and is regulated by geranylgeranyl pyrophosphate

Prenylation (or geranylgeranylation) of Rab GTPases is catalysed by RGGT (Rab geranylgeranyl transferase) and requires REP (Rab escort protein). In the classical pathway, REP associates first with unprenylated Rab, which is then prenylated by RGGT. In the alternative pathway, REP associates first with RGGT; this complex then binds and prenylates Rab proteins. In the present paper we show that REP mutants defective in RGGT binding (REP1 F282L and REP1 F282L/V290F) are unable to compete with wild-type REP in the prenylation reaction in vitro. When over-expressed in cells, REP wild-type and mutants are unable to form stable cytosolic complexes with endogenous unprenylated Rabs. These results suggest that the alternative pathway may predominate in vivo. We also extend previous suggestions that GGPP (geranylgeranyl pyrophosphate) acts as an allosteric regulator of the prenylation reaction. We observed that REP-RGGT complexes are formed in vivo and are unstable in the absence of intracellular GGPP. RGGT increases the ability of REP to extract endogenous prenylated Rabs from membranes in vitro by stabilizing a soluble REP-RGGT-Rab-GG (geranylgeranylated Rab) complex. This effect is regulated by GGPP, which promotes the dissociation of RGGT and REP-Rab-GG to allow delivery of prenylated Rabs to membranes.     

The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.

C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines. We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus. Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain. The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane. Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants. Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S. cerevisiae.     

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.