The Small GTPase Rab5c Exerts Bi-Function in Singapore Grouper Iridovirus Infections and Cellular Responses in the Grouper, Epinephelus coioides.

The small GTPase Rab5 is one of the master regulators of vesicular trafficking that participates in early stages of the endocytic pathway, such as endocytosis and endosome maturation. Three Rab5 isoforms (a, b, and c) share high sequence identity, and exhibit complex functions. However, the role of Rab5c in virus infection and cellular immune responses remains poorly understood. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected grouper spleen (GS) cells, we investigated the role of Rab5c in virus infection and host immune responses. Rab5c was cloned from the orange-spotted grouper, Epinephelus coioides, and termed EcRab5c. EcRab5c encoded a 220-amino-acid polypeptide, showing 99% and 91% identity to Anabas testudineus, and Homo sapiens, respectively. Confocal imaging showed that EcRab5c localized as punctate structures in the cytoplasm. However, a constitutively active (CA) EcRab5c mutant led to enlarged vesicles, while a dominant negative (DN) EcRab5c mutant reduced vesicle structures. EcRab5c expression levels were significantly increased after SGIV infection. EcRab5c knockdown, or CA/DN EcRab5c overexpression significantly inhibited SGIV infection. Using single-particle imaging analysis, we further observed that EcRab5c disruption impaired crucial events at the early stage of SGIV infection, including virus binding, entry, and transport from early to late endosomes, at the single virus level. Furthermore, it is the first time to investigate that EcRab5c is required in autophagy. Equally, EcRab5c positively regulated interferon-related factors and pro-inflammatory cytokines. In summary, these data showed that EcRab5c exerted a bi-functional role on iridovirus infection and host immunity in fish, which furthers our understanding of virus and host immune interactions.

Rac1-dependent endocytosis and Rab5-dependent intracellular trafficking are required by Enterovirus A71 and Coxsackievirus A10 to establish infections.

Enterovirus A71 (EVA71) and Coxsackievirus A10 (CVA10) are representative types of Enterovirus A. Dependent on the host cell types, the EVA71 entry may utilize clathrin-, caveola-, and endophilin-A2-mediated endocytosis. However, the cell-entry and intracellular trafficking pathways of CVA10, using KREMEN1 as its receptor, are unclear. Here, we tested the relevant mechanisms through RNA interference (RNAi) and chemical inhibitors. We found that endocytosis of EVA71 and CVA10 in rhabdomyosarcoma (RD) cells engaged multiple pathways, and both viruses required Rac1. Interestingly, while CDC42 and Pak1 participated in EVA71 infection, PI3K played a role in CVA10 infection. The functions of Rab proteins in intracellular trafficking of CVA10 and EVA71 were examined by RNAi. Knockdown of Rab5 and Rab21 significantly reduced CVA10 infectivity, while knockdown of Rab5, Rab7 and Rab9 reduced EVA71 infectivity. Confocal microscopy confirmed the colocalization of CVA10 virions with Rab5 or Rab21, and colocalization of EVA71 virions with Rab5 or Rab7. Additionally, we observed that both CVA10 and EVA71 infections were inhibited by endosome acidification inhibitors, bafilomycin-A1 and NH4Cl. Together, our findings comparatively illustrate the entry and intracellular trafficking processes of representative Enterovirus A types and revealed novel enterovirus intervention targets.

RAB5A and TRAPPC6B are novel targets for Shiga toxin 2a inactivation in kidney epithelial cells.

The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells. In EHEC pathogenesis, the disturbance of the kidney epithelium by Stx becomes increasingly recognised, but how this exactly occurs is unknown. To explore this molecularly, we investigated the Stx receptor content and transcriptomic profile of two human renal epithelial cell lines: highly Stx-sensitive ACHN cells and largely Stx-insensitive Caki-2 cells. Though both lines exhibited the Stx receptor globotriaosylceramide, RNAseq revealed strikingly different transcriptomic responses to an Stx challenge. Using RNAi to silence factors involved in ACHN cells' Stx response, the greatest protection occurred when silencing RAB5A and TRAPPC6B, two host factors that we newly link to Stx trafficking. Silencing these factors alongside YKT6 fully prevented the cytotoxic Stx effect. Overall, our approach reveals novel subcellular targets for potential therapies against Stx-mediated kidney failure.

The Mevalonate Pathway Is a Druggable Target for Vaccine Adjuvant Discovery.

Motivated by the clinical observation that interruption of the mevalonate pathway stimulates immune responses, we hypothesized that this pathway may function as a druggable target for vaccine adjuvant discovery. We found that lipophilic statin drugs and rationally designed bisphosphonates that target three distinct enzymes in the mevalonate pathway have potent adjuvant activities in mice and cynomolgus monkeys. These inhibitors function independently of conventional "danger sensing." Instead, they inhibit the geranylgeranylation of small GTPases, including Rab5 in antigen-presenting cells, resulting in arrested endosomal maturation, prolonged antigen retention, enhanced antigen presentation, and T cell activation. Additionally, inhibiting the mevalonate pathway enhances antigen-specific anti-tumor immunity, inducing both Th1 and cytolytic T cell responses. As demonstrated in multiple mouse cancer models, the mevalonate pathway inhibitors are robust for cancer vaccinations and synergize with anti-PD-1 antibodies. Our research thus defines the mevalonate pathway as a druggable target for vaccine adjuvants and cancer immunotherapies.

Contractility, differential tension and membrane removal lead zebrafish epiboly biomechanics.

Precise tissue remodeling during development is essential for shaping embryos and optimal organ function. Epiboly is an early gastrulation event by which the blastoderm expands around the yolk to engulf it. Three different layers are involved in this process, an epithelial layer (the enveloping layer, EVL), the embryo proper, constituted by the deep cells (DCs), and the yolk cell. Although teleost epiboly has been studied for many years, a clear understanding of its mechanics was still missing. Here we present new information on the cellular, molecular and mechanical elements involved in epiboly that, together with some other recent data and upon comparison with previous biomechanical models, lets conclude that the expansion of the epithelia is passive and driven by active cortical contraction and membrane removal in the adjacent layer, the External Yolk Syncytial Layer (E-YSL). The isotropic actomyosin contraction of the E-YSL cortex generates an anisotropic stress pattern and a directional net movement consequence of the differences in the deformation response of the 2 opposites adjacent domains (EVL and the Yolk Cytoplasmic Layer - YCL). Contractility is accompanied by the local formation of membrane folds and its removal by Rab5ab dependent macropinocytosis. The increase in area of the epithelia during the expansion is achieved by cell-shape changes (flattening) responding to spherical geometrical cues. The counterbalance between the geometry of the embryo and forces dissipation among different elements is therefore essential for epiboly global coordination.

Rab5a­mediated autophagy regulates the phenotype and behavior of vascular smooth muscle cells.

Rab5a, a key member of the Rab family of GTPases, was determined to be a regulator of vascular smooth muscle cell (VSMC) proliferation and migration. However, the exact regulatory mechanism remains unclear. As Rab5a has been shown to be associated with autophagy, which is essential for the conversion of VSMCs from a contractile to a synthetic phenotype in order to prevent cell death due to oxidative stress. The present study hypothesized that autophagy may be responsible for the proliferation and migration of VSMCs via the Rab5a protein. The aim of the present study was to evaluate the effect of Rab5a on autophagy in VSMCs. The human aorta vascular smooth muscle cell line, T/G HA­VSMCs, was treated with small interfering (si)RNA against Rab5a and/or platelet­derived growth factor (PDGF). Following treatment, the phenotype transition of the VSMCs was evaluated by detecting the mRNA and protien expression levels of VSMC molecular markers using reverse transcription­quantitative polymerase chain reaction and western blotting, respectively. In addition, autophagy in VSMCs was evaluated by western blotting for autophagy­associated proteins, flow cytometry of acidic vesicular organelles, punctate fluorescence of microtubule associated protein light chain 3 and transmission electron microscopy of typical scattered double­membrane vacuolar structures. Additionally, the proliferation, migration, cell cycle and apoptotic response of VSMCs were detected by sulforhodamine B assay, transwell assay and flow cytometry, respectively. The results revealed that transfection with siRNA against Rab5a led to a significant decrease in Rab5a protein expression, while the reduced expression trend of Rab5a was rescued by intervention with PDGF. Furthermore, cells transfected with siRNA against Rab5a inhibited the autophagy of VSMCs. Downregulated Rab5a inhibited the phenotype transition of VSMCs. Additionally, downregulated Rab5a led to slowed cell growth, decreased numbers of migrated cells, decreased numbers of cells at the G0­G1 phase and a higher apoptosis rate. However, PDGF significantly rescued these phenomena caused by siRNA against Rab5a. These results indicated that Rab5a­mediated autophagy may regulate the phenotype transition and cell behavior of VSMCs through the activation of the extracellular­regulated kinase 1/2 signaling pathway.

Osteoblast regulation via ligand-activated nuclear trafficking of the oxytocin receptor.

We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr-EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with ß-arrestins (Arrbs), the small GTPase Rab5, importin-ß (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.

Rab5a is required for spindle length control and kinetochore-microtubule attachment during meiosis in oocytes.

Rab GTPases are highly conserved components of vesicle trafficking pathways. Rab5, as a master regulator of endocytic trafficking, has been shown to function in membrane tethering and docking. However, the function of Rab5 in meiosis has not been addressed. Here, we report elongated spindles and misaligned chromosomes, with kinetochore-microtubule misattachments, on specific depletion of Rab5a in mouse oocytes. Moreover, the localization and levels of centromere protein F (CENPF), a component of the nuclear matrix, are severely reduced at kinetochores in metaphase oocytes following Rab5a knockdown. Consistent with this finding, nuclear lamina disassembly in the transition from prophase arrest to meiosis I is also impaired in Rab5a-depleted oocytes. Notably, oocyte-specific ablation of CENPF phenocopies the meiotic defects resulting from Rab5a knockdown. In summary, our data support a model where Rab5a-positive vesicles, likely through interaction with nuclear lamina, modulate CENPF localization and levels at centromeres, consequently ensuring proper spindle length and kinetochore-microtubule attachment in meiotic oocytes.

Rab5 isoforms orchestrate a "division of labor" in the endocytic network; Rab5C modulates Rac-mediated cell motility.

Rab5, the prototypical Rab GTPase and master regulator of the endocytic pathway, is encoded as three differentially expressed isoforms, Rab5A, Rab5B and Rab5C. Here, we examined the differential effects of Rab5 isoform silencing on cell motility and report that Rab5C, but neither Rab5A nor Rab5B, is selectively associated with the growth factor-activation of Rac1 and with enhanced cell motility. Initial observations revealed that silencing of Rab5C expression, but neither Rab5A nor Rab5C, led to spindle-shaped cells that displayed reduced formation of membrane ruffles. When subjected to a scratch wound assay, cells depleted of Rab5C, but not Rab5A or Rab5B, demonstrated reduced cell migration. U937 cells depleted of Rab5C also displayed reduced cell motility in a Transwell plate migration assay. To examine activation of Rac, HeLa cells stably expressing GFP-Rac1 were independently depleted of Rab5A, Rab5B or Rab5C and seeded onto coverslips imprinted with a crossbow pattern. 3-D GFP-Rac1 images of micro-patterned cells show that GFP-Rac1 was less localized to the cell periphery in the absence of Rab5C. To confirm the connection between Rab5C and Rac activation, HeLa cells depleted of Rab5 isoforms were starved and then stimulated with EGF. Rac1 pull-down assays revealed that EGF-stimulated Rac1 activity was significantly suppressed in Rab5C-suppressed cells. To determine whether events upstream of Rac activation were affected by Rab5C, we observed that EGF-stimulated Akt phosphorylation was suppressed in cells depleted of Rab5C. Finally, since spatio-temporal assembly/disassembly of adhesion complexes are essential components of cell migration, we examined the effect of Rab5 isoform depletion on the formation of focal adhesion complexes. Rab5C-depleted HeLa cells have significantly fewer focal adhesion foci, in accordance with the lack of persistent lamellipodial protrusions and reduced directional migration. We conclude that Rab5 isoforms selectively oversee the multiple signaling and trafficking events associated with the endocytic network.

A specific subset of RabGTPases controls cell surface exposure of MT1-MMP, extracellular matrix degradation and three-dimensional invasion of macrophages.

The matrix metalloproteinase MT1-MMP has a major impact on invasive cell migration in both physiological and pathological settings such as immune cell extravasation or metastasis of cancer cells. Surface-associated MT1-MMP is able to cleave components of the extracellular matrix, which is a prerequisite for proteolytic invasive migration. However, current knowledge on the molecular mechanisms that regulate MT1-MMP trafficking to and from the cell surface is limited. We have identified three members of the RabGTPase family, Rab5a, Rab8a and Rab14, as crucial regulators of MT1-MMP trafficking and function in primary human macrophages. Both overexpressed and endogenous forms show prominent colocalisation with MT1-MMP-positive vesicles, whereas expression of mutant constructs, as well as siRNA-induced knockdown, reveal that these RabGTPases are crucial in the regulation of MT1-MMP surface exposure, contact of MT1-MMP-positive vesicles with podosomes, extracellular matrix degradation in two and three dimensions, as well as three-dimensional proteolytic invasion of macrophages. Collectively, our results identify Rab5a, Rab8a and Rab14 as major regulators of MT1-MMP trafficking and invasive migration of primary human macrophages, which could be promising potential targets for manipulation of immune cell invasion.

Modulation of foot-and-mouth disease virus pH threshold for uncoating correlates with differential sensitivity to inhibition of cellular Rab GTPases and decreases infectivity in vivo.

The role of cellular Rab GTPases that govern traffic between different endosome populations was analysed on foot-and-mouth disease virus (FMDV) infection. Changes of viral receptor specificity did not alter Rab5 requirement for infection. However, a correlation between uncoating pH and requirement of Rab5 for infection was observed. A mutant FMDV with less acidic uncoating pH threshold was less sensitive to inhibition of Rab5, whereas another mutant with more acidic requirements was more sensitive to inhibition of Rab5. On the contrary, opposed correlations between uncoating pH and dependence of Rab function were observed upon expression of dominant-negative forms of Rab7 or 11. Modulation of uncoating pH also reduced FMDV virulence in suckling mice. These results are consistent with FMDV uncoating inside early endosomes and indicate that displacements from optimum pH for uncoating reduce viral fitness in vivo.

Functional CSF-1 receptors are located at the nuclear envelope and activated via the p110δ isoform of PI 3-kinase.

Colony stimulating factor-1 (CSF-1) and its receptor (CSF-1R) are key regulators of macrophage biology, and their elevated expression in cancer cells has been linked to poor prognosis. CSF-1Rs are thought to function at the plasma membrane. We show here that functional CSF-1Rs are present at the nuclear envelope of various cell types, including primary macrophages, human cancer cell lines, and primary human carcinomas. In response to CSF-1, added to intact cells or isolated nuclei, nucleus-associated CSF-1R became phosphorylated and triggered the phosphorylation of Akt and p27 inside the nucleus. Extracellularly added CSF-1 was also found to colocalize with nucleus-associated CSF-1Rs. All these activities were found to depend selectively on the activity of the p110δ isoform of phosphoinositide 3-kinase (PI3K). This finding was related to the p110δ-dependent translocation of exogenous CSF-1 to the nucleus-associated CSF-1Rs, correlating with a prominent role of p110δ in activation of the Rab5 GTPase, a key regulator of the endocytic trafficking. siRNA-silencing of Rab5a phenocopied p110δ inactivation and nuclear CSF-1 signaling. Our work demonstrates for the first time the presence of functional nucleus-associated CSF-1Rs, which are activated by extracellular CSF-1 by a mechanism that involves p110δ and Rab5 activity. These findings may have important implications in cancer development.

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking.

The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

Small GTPase Rab5 participates in chromosome congression and regulates localization of the centromere-associated protein CENP-F to kinetochores.

Rab5 is a small GTPase known to regulate vesicular trafficking during interphase. Here, we show that Rab5 also plays an unexpected role during mitotic progression. RNAi-mediated silencing of Rab5 caused defects in chromosome congression and extensive prometaphase delay, and it correlated with a severe reduction in the localization of the centromere-associated protein CENP-F to kinetochores. CENP-F is a component of the nuclear matrix required for chromosome congression that, at mitotic entry, localizes to the nuclear envelope and assembles on kinetochores, contributing to the establishment of kinetochore microtubule interactions. We found that Rab5 forms a complex with a subset of CENP-F in mitotic cells and regulates the kinetics of release of CENP-F from the nuclear envelope and its accumulation on kinetochores. Simultaneous depletion of both Rab5 and CENP-F recapitulated the mitotic defects caused by silencing of either Rab5 or CENP-F alone, indicating epistatic roles for these two proteins in the pathway that orchestrates chromosome congression. These results reveal the involvement of Rab5 in the proper execution of mitotic programs whose deregulation can undermine chromosomal stability.

Rab5 GTPase controls chromosome alignment through Lamin disassembly and relocation of the NuMA-like protein Mud to the poles during mitosis.

The small GTPase Rab5 is a conserved regulator of membrane trafficking; it regulates the formation of early endosomes, their transport along microtubules, and the fusion to the target organelles. Although several members of the endocytic pathway were recently implicated in spindle organization, it is unclear whether Rab5 has any role during mitosis. Here, we describe that Rab5 is required for proper chromosome alignment during Drosophila mitoses. We also found that Rab5 associated in vivo with nuclear Lamin and mushroom body defect (Mud), the Drosophila counterpart of nuclear mitotic apparatus protein (NuMA). Consistent with this finding, Rab5 was required for the disassembly of the nuclear envelope at mitotic entry and the accumulation of Mud at the spindle poles. Furthermore, Mud depletion caused chromosome misalignment defects that resembled the defects of Rab5 RNAi cells, and double-knockdown experiments indicated that the two proteins function in a linear pathway. Our results indicate a role for Rab5 in mitosis and reinforce the emerging view of the contributions made by cell membrane dynamics to spindle function.

Coordination of the Rab5 cycle on macropinosomes.

The GTPase Rab5a regulates the homotypic and heterotypic fusion of membranous organelles during the early stages of endocytosis. Many of the molecules which regulate the Rab5a cycle of association with membranes, activation, deactivation and dissociation are known. However, the extent to which these molecular scale activities are coordinated on membranes to affect the behavior of individual organelles has not been determined. This study used novel Förster resonance energy transfer (FRET) microscopic methods to analyze the Rab5a cycle on macropinosomes, which are large endocytic vesicles that form in ruffled regions of cell membranes. In Cos-7 cells and mouse macrophages stimulated with growth factors, Rab5a activation followed immediately after its recruitment to newly formed macropinosomes. Rab5a activity increased continuously and uniformly over macropinosome membranes then decreased continuously, with Rab5a deactivation preceding dissociation by 1-12 min. Although the maximal levels of Rab5a activity were independent of organelle size, Rab5a cycles were longer on larger macropinosomes, consistent with an integrative activity governing Rab5a dynamics on individual organelles. The Rab5a cycle was destabilized by microtubule depolymerization and by bafilomycin A1. Overexpression of activating and inhibitory proteins indicated that active Rab5a stabilized macropinosomes. Thus, overall Rab5a activity on macropinosomes is coordinated by macropinosome structure and physiology.

Rab5 and class III phosphoinositide 3-kinase Vps34 are involved in hepatitis C virus NS4B-induced autophagy.

Autophagy has been shown to facilitate replication or production of hepatitis C virus (HCV); nevertheless, how HCV induces autophagy remains unclear. Here, we demonstrate that HCV nonstructural protein 4B (NS4B) alone can induce autophagy signaling; amino acid residues 1 to 190 of NS4B are sufficient for this induction. Further studies showed that the phosphorylation levels of S6K and 4E-BP1 were not altered, suggesting that the mTOR/S6 kinase pathway and mTOR/4E-BP1 pathway did not contribute to NS4B- or HCV-induced autophagy. Inhibition of Rab5 function by silencing Rab5 or overexpressing dominant-negative Rab5 mutant (S34N) resulted in significant reduction of NS4B- or HCV-induced autophagic vesicle formation. Moreover, the autophagy induction was impaired by inhibition of class III phosphoinositide 3-kinase (PI 3-kinase) Vps34 function. Finally, the coimmunoprecipitation assay indicated that NS4B formed a complex with Rab5 and Vps34, supporting the notion that Rab5 and Vps34 are involved in NS4B-induced autophagy. Taken together, these results not only reveal a novel role of NS4B in autophagy but also offer a clue to the mechanism of HCV-induced autophagy.

Cannabinoid receptor 2 undergoes Rab5-mediated internalization and recycles via a Rab11-dependent pathway.

Cannabinoid receptor 2 (CB2) is a GPCR highly expressed on the surface of cells of the immune system, supporting its role in immunomodulation. This study has investigated the trafficking properties of this receptor when stably expressed by HEK-293 cells. As previously reported, cell surface CB2 rapidly internalized upon exposure to agonist. Direct evidence of CB2 recycling was observed upon competitive removal of the stimulating agonist by inverse agonist. CB2 also underwent slow constitutive internalization when agonist was absent and was up-regulated in the presence of inverse agonist. Co-expression of CB2 and dominant negative Rab5 resulted in a significantly reduced capacity for receptors to internalize with no effect on recycling of the internalized receptors. Conversely, co-expression with dominant negative Rab11 did not alter the ability of CB2 to internalize but did impair their ability to return to the cell surface. Co-expression of wild-type, dominant negative or constitutively active Rab4 with CB2 did not alter basal surface expression, extent of internalization, or extent of recycling. These results suggest that Rab5 is involved in CB2 endocytosis and that internalized receptors are recycled via a Rab11 associated pathway rather than the rapid Rab4 associated pathway. This report provides the first comprehensive description of CB2 internalization and recycling to date.

Non-SCF-type F-box protein Roy1/Ymr258c interacts with a Rab5-like GTPase Ypt52 and inhibits Ypt52 function.

Skp1/Cul1/F-box (SCF)-type F-box proteins are a component of the Cullin-RING SCF ubiquitin E3 ligase, which is involved in numerous cellular processes. However, the function of non-SCF-type F-box proteins remains largely unknown. The Rab5-like small guanosine 5'-triphosphatase Vps21/Ypt51 is a key regulator of intracellular transportation; however, deletion of its isoforms, Ypt52 and Ypt53, results in only a modest inhibition of intracellular trafficking. The function of these proteins therefore remains largely elusive. Here we analyze the role of a previously uncharacterized non-SCF-type F-box protein, Roy1/Ymr258c, in cell growth and intracellular transport in Saccharomyces cerevisiae. Roy1 binds to Ypt52 under physiological conditions, and Skp1 is indispensable for the association of Roy1 with Ypt52. The vps21Δ yeast cells exhibit severe deficiencies in cell growth and intracellular trafficking, whereas simultaneous deletion of roy1 alleviates the defects caused by deletion of vps21. However, additional disruption of ypt52 in roy1Δvps21Δ cells largely suppresses the cell growth and trafficking observed in roy1Δvps21Δ cells. We demonstrate that Roy1 interacts with guanosine 5'-diphosphate-bound and nucleotide-free Ypt52 and thereby inhibits the formation of guanosine 5'-triphosphate-bound, active Ypt52. These results thus indicate that Roy1 negatively modulates cell viability and intracellular transport by suppressing Ypt52.