Disorder of trafficking system of plasma membrane and vacuole antiporter proteins causes hypersensitive response to salinity stress in <Emphasis Type="Italic">Arabidopsis Thaliana</Emphasis>

Rab GTPases play an important role in regulating intracellular vesicular trafficking in eukaryotic cells. Previously, we found that Oryza sativa rice Rab11 (OsRab11) is required for the regulation of vesicular trafficking from the trans- Golgi network (TGN) to the plasma membrane (PM) and/or vacuoles. To further elucidate the relationship between vesicular trafficking and abiotic and biotic stresses, we determined OsRab11 expression levels under several environmental stress conditions. OsRab11 expression was induced by pathogens, jasmonic acid (JA), and high salt treatment. Under high salt conditions, dominant negative OsRab11(S28N) mutant plants exhibited a hypersensitive phenotype similar to that of sos1-1, whereas overexpressed-OsRab11 plants showed resistance to high salt stress. When the expression of vacuolar and PM Na⁺/H⁺ antiporter genes such as AtNHX1, AtNHX2, and AtSOS1 was examined, there was no significant difference between the wild-type and OsRab11(S28N) mutant plants. However, PM trafficking of AtSOS1-green fluorescent protein (GFP) in 35S::AtSOS1-GFP sos1-1 plants was severely impaired by T7-OsRab11(S28N) expression. Similarly, vacuolar trafficking of AtNHX2-GFP was inhibited by T7-OsRab11 (S28N) expression. These results indicate that trafficking of PM and vacuolar antiporter proteins by OsRab11 is important for high salt stress resistance.     

Overexpression of Constitutively Active OsRab11 in Plants Enhances Tolerance to High Salinity Levels

Rab proteins are key regulators of intracellular trafficking between specific compartments in a cell. Among them, Rab11, a widely conserved sub-group, mainly regulates plasma membrane (PM) trafficking. Previously, we reported that Oryza sativa Rab11 (OsRab11) plays an important role in the intracellular trafficking from the trans-Golginetwork (TGN) to the plasma membrane (PM) and prevacuolar compartments (PVCs), and in the plant’s response to high salt stress. In this study, when the constitutively active mutant of OsRab11, (CA OsRab11(Q73L)) was co-transformed with Arabidopsis Ca²⁺-ATPase8-GFP (ACA8-GFP) or sporamin-GFP (Spo-GFP) into Arabidopsis protoplasts, the PM or vacuolar trafficking proportion of the reporter proteins was highly increased. Transgenic Arabidopsis plants overexpressing (OE) CA OsRab11(Q73L) exhibited enhanced tolerance to high salt stress and exogenous abscisic acid (ABA) compared to Col plants. Moreover, certain stress-responsive genes were expressed under high salt stress and ABA treatment in OEOsRab11(Q73L) plants. Thus, these results suggest that the active conformation of OsRab11 may be required to modulate plant responses to salt and ABA via the regulation of the expression of stress-responsive genes.     

Molecular and biochemical analyses of OsRab7, a rice Rab7 homolog

Rab7 is a small GTP-binding protein important in early to late endosome/lysosome vesicular transport in mammalian cells. We have isolated a Rab7 cDNA clone, OsRab7, from a cold-treated rice cDNA library by the subtraction screening method. The cDNA encodes a polypeptide of 206 amino acids with a calculated molecular mass of about 23 kDa. Its predicted amino acid sequence shows significantly high identity with the sequences of other Rab7 proteins. His-tagged OsRab7 bound to radiolabeled GTPgammaS in a specific and stoichiometric manner. Biochemical and structural properties of the Rab7 wild type (WT) protein were compared to those of Q67L and T22N mutants. The detergent 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS) increased the guanine nucleotide binding and hydrolysis activities of Rab7WT. The OsRab7Q67L mutant showed much lower GTPase activity compared to the WT protein untreated with CHAPS, and the T22N mutant showed no GTP binding activity at all. The OsRab7Q67L mutant was constitutively active for guanine nucleotide binding while the T22N mutant (dominant negative) showed no guanine nucleotide binding activity. When bound to GTP, the Rab7WT and the Q67L mutants were protected from tryptic proteolysis. The cleavage pattern of the Rab7T22N mutant, however, was not affected by GTP addition. Northern and Western blot analyses suggested that OsRab7 is distributed in various tissues of rice. Furthermore, expression of a rice Rab7 gene was differentially regulated by various environmental stimuli such as cold, NaCl, dehydration, and ABA. In addition, subcellular localization of OsRab7 was investigated in the Arabidopsis protoplasts by a double-labeling experiment using GFP-fused OsRab7 and FM4-64. GFP-OsRab7 is localized to the vacuolar membrane, suggesting that OsRab7 is implicated in a vesicular transport to the vacuole in plant cells.     

Rab Family Proteins Regulate the Endosomal Trafficking and Function of RGS4

RGS4, a heterotrimeric G-protein inhibitor, localizes to plasma membrane (PM) and endosomal compartments. Here, we examined Rab-mediated control of RGS4 internalization and recycling. Wild type and constitutively active Rab5 decreased RGS4 PM levels while increasing its endosomal targeting. Rab5, however, did not appreciably affect the PM localization or function of the M1 muscarinic receptor (M1R)/G_q signaling cascade. RGS4-containing endosomes co-localized with subsets of Rab5-, transferrin receptor-, and Lamp1/Lysotracker-marked compartments suggesting RGS4 traffics through PM recycling or acidified endosome pathways. Rab7 activity promoted TGN association, whereas Rab7(dominant negative) trapped RGS4 in late endosomes. Furthermore, RGS4 was found to co-localize with an endosomal pool marked by Rab11, the protein that mediates recycling/sorting of proteins to the PM. The Cys-12 residue in RGS4 appeared important for its Rab11-mediated trafficking to the PM. Rab11(dominant negative) decreased RGS4 PM levels and increased the number of RGS4-containing endosomes. Inhibition of Rab11 activity decreased RGS4 function as an inhibitor of M1R activity without affecting localization and function of the M1R/G_q signaling complex. Thus, both Rab5 activation and Rab11 inhibition decreased RGS4 function in a manner that is independent from their effects on the localization and function of the M1R/G_q signaling complex. This is the first study to implicate Rab GTPases in the intracellular trafficking of an RGS protein. Thus, Rab GTPases may be novel molecular targets for the selective regulation of M1R-mediated signaling via their specific effects on RGS4 trafficking and function.     

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella‐containing vacuole

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high‐throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.     

Arf1 orchestrates Rab GTPase conversion at the trans-Golgi network

Rab family GTPases are key organizers of membrane trafficking and function as markers of organelle identity. Accordingly, Rab GTPases often occupy specific membrane domains, and mechanisms exist to prevent the inappropriate mixing of distinct Rab domains. The yeast Golgi complex can be divided into two broad Rab domains: Ypt1 (Rab1) and Ypt6 (Rab6) are present at the early/medial Golgi and sharply transition to Ypt31/32 (Rab11) at the late Golgi/trans-Golgi network (TGN). This Rab conversion has been attributed to GTPase-activating protein (GAP) cascades in which Ypt31/32 recruits the Rab-GAPs Gyp1 and Gyp6 to inactivate Ypt1 and Ypt6, respectively. Here we report that Rab transition at the TGN involves additional layers of regulation. We provide new evidence confirming the TRAPPII complex as an important regulator of Ypt6 inactivation and uncover an unexpected role of the Arf1 GTPase in recruiting Gyp1 to drive Ypt1 inactivation at the TGN. Given its established role in directly recruiting TRAPPII to the TGN, Arf1 is therefore a master regulator of Rab conversion on maturing Golgi compartments.     

EPI64 interacts with Slp1/JFC1 to coordinate Rab8a and Arf6 membrane trafficking

Cell function requires the integration of cytoskeletal organization and membrane trafficking. Small GTP-binding proteins are key regulators of these processes. We find that EPI64, an apical microvillar protein with a Tre-2/Bub2/Cdc16 (TBC) domain that stabilizes active Arf6 and has RabGAP activity, regulates Arf6-dependent membrane trafficking. Expression of EPI64 in HeLa cells induces the accumulation of actin-coated vacuoles, a distinctive phenotype seen in cells expressing constitutively active Arf6. Expression of EPI64 with defective RabGAP activity does not induce vacuole formation. Coexpression of Rab8a suppresses the vacuole phenotype induced by EPI64, and EPI64 expression lowers the level of Rab8-GTP in cells, strongly suggesting that EPI64 has GAP activity toward Rab8a. JFC1, an effector for Rab8a, colocalizes with and binds directly to a C-terminal region of EPI64. Together this region and the N-terminal TBC domain of EPI64 are required for the accumulation of vacuoles. Through analysis of mutants that uncouple JFC1 from either EPI64 or from Rab8-GTP, our data suggest a model in which EPI64 binds JFC1 to recruit Rab8a-GTP for deactivation by the RabGAP activity of EPI64. We propose that EPI64 regulates membrane trafficking both by stabilizing Arf6-GTP and by inhibiting the recycling of membrane through the tubular endosome by decreasing Rab8a-GTP levels.     

Rab8 regulates basolateral secretory, but not recycling, traffic at the recycling endosome

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the mu1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.     

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.     

A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum‐derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system‐mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans‐Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase‐activating Protein (GAP) ACAP1 to dysregulate Arf6‐/Rab8a‐dependent transport within the recycling endosome, which resulted in accretion of TGN‐associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.     

Arf GTPase-activating protein ASAP1 interacts with Rab11 effector FIP3 and regulates pericentrosomal localization of transferrin receptor-positive recycling endosome

ADP-ribosylation factors (Arfs) and Arf GTPase-activating proteins (GAPs) are key regulators of membrane trafficking and the actin cytoskeleton. The Arf GAP ASAP1 contains an N-terminal BAR domain, which can induce membrane tubulation. Here, we report that the BAR domain of ASAP1 can also function as a protein binding site. Two-hybrid screening identified FIP3, which is a putative Arf6- and Rab11-effector, as a candidate ASAP1 BAR domain-binding protein. Both coimmunoprecipitation and in vitro pulldown assays confirmed that ASAP1 directly binds to FIP3 through its BAR domain. ASAP1 formed a ternary complex with Rab11 through FIP3. FIP3 binding to the BAR domain stimulated ASAP1 GAP activity against Arf1, but not Arf6. ASAP1 colocalized with FIP3 in the pericentrosomal endocytic recycling compartment. Depletion of ASAP1 or FIP3 by small interfering RNA changed the localization of transferrin receptor, which is a marker of the recycling endosome, in HeLa cells. The depletion also altered the trafficking of endocytosed transferrin. These results support the conclusion that ASAP1, like FIP3, functions as a component of the endocytic recycling compartment.     

Differential Effects of TBC1D15 and Mammalian Vps39 on Rab7 Activation State,Lysosomal Morphology,and Growth Factor Dependence

The small GTPase Rab7 promotes fusion events between late endosomes and lysosomes. Rab7 activity is regulated by extrinsic signals, most likely via effects on its guanine nucleotide exchange factor (GEF) or GTPase-activating protein (GAP). Based on their homology to the yeast proteins that regulate the Ypt7 GTP binding state, TBC1D15, and mammalian Vps39 (mVps39) have been suggested to function as the Rab7 GAP and GEF, respectively. We developed an effector pull-down assay to test this model. TBC1D15 functioned as a Rab7 GAP in cells, reducing Rab7 binding to its effector protein RILP, fragmenting the lysosome, and conferring resistance to growth factor withdrawal-induced cell death. In a cellular context, TBC1D15 GAP activity was selective for Rab7. TBC1D15 overexpression did not inhibit transferrin internalization or recycling, Rab7-independent processes that require Rab4, Rab5, and Rab11 activation. TBC1D15 was thus renamed Rab7-GAP. Contrary to expectations for a Rab7 GEF, mVps39 induced lysosomal clustering without increasing Rab7 GTP binding. Moreover, a dominant-negative mVps39 mutant fragmented the lysosome and promoted growth factor independence without decreasing Rab7-GTP levels. These findings suggest that a protein other than mVps39 serves as the Rab7 GEF. In summary, although only TBC1D15/Rab7-GAP altered Rab7-GTP levels, both Rab7-GAP and mVps39 regulate lysosomal morphology and play a role in maintaining growth factor dependence.     

The GTPase-activating enzyme Gyp1p is required for recycling of internalized membrane material by inactivation of the Rab/Ypt GTPase Ypt1p

Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments. A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo. Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material. Interestingly, recycling of GFP-Snc1p in gyp1 Delta cells is partially restored by reducing the activity of Ypt1p. Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling. Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment. Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material.     

Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells

To maintain polarity, epithelial cells continuously sort transmembrane proteins to the apical or basolateral membrane domains during biosynthetic delivery or after internalization. During biosynthetic delivery, some cargo proteins move from the trans-Golgi network (TGN) into recycling endosomes (RE) before being delivered to the plasma membrane. However, proteins that regulate this transport step remained elusive. In this study, we show that Rab13 partially colocalizes with TGN38 at the TGN and transferrin receptors in RE. Knockdown of Rab13 with short hairpin RNA in human bronchial epithelial cells or overexpression of dominant-active or dominant-negative alleles of Rab13 in Madin-Darby canine kidney cells disrupts TGN38/46 localization at the TGN. Moreover, overexpression of Rab13 mutant alleles inhibits surface arrival of proteins that move through RE during biosynthetic delivery (vesicular stomatitis virus glycoprotein [VSVG], A-VSVG, and LDLR-CT27). Importantly, proteins using a direct route from the TGN to the plasma membrane are not affected. Thus, Rab13 appears to regulate membrane trafficking between TGN and RE.     

Plant PRA plays an important role in intracellular vesicular trafficking between compartments as GDF

Rab GTPases like Ras-related monomeric GTPases are well known to regulate intracellular vesicle trafficking by cycling between membrane-bound and cytosolic states. The functions of these proteins are controlled by upstream regulators and downstream effectors. Ypt/Rabs transmit signals to downstream effectors in a GTP-dependent manner. GDP-bound Rab proteins are extracted from their target membrane by cytosolic proteins known as GDP dissociation inhibitors (GDIs), and the Rab GTPase is recruited to the membrane compartment following dissociation from the GDI by GDI displacement factor (GDF). Now, we''re going to discuss the role of plant PRA concerted with Rab and GDI proteins by recycling Rab between membrane and cytosol for intracellular trafficking of cargo proteins.Key words: GDF, GDI, PRA1, Rab, vacuolar trafficking, vesicle traffickingAlthough Rabs appear to undergo multiple cycles of GDI-mediated delivery to, and extraction from membranes,¹ the mechanisms underlying Rab membrane delivery and association by GDI and other factors remain unclear. GDP-GTP exchange occurs at the target membrane, catalyzed by a guanine nucleotide exchange factor (GEF),^2,3 and the GTP-bound Rab transmits signals to downstream effectors and associates with the membrane to ensure proper docking and fusion of transport vesicles.⁴ After vesicle fusion on its target membrane, subsequently hydrolysis of GTP by the Rab is facilitated by GTPase-activating proteins (GAPs).⁴ The resulting GDP-bound Rab is subsequently retrieved from the membrane by GDI, which then maintains Rab in the cytosol to complete the cycle.Many research groups isolated PRAs, a homolog of human YIP3, in several two-hybrid screenings as interacting with multiple Rabs in their GTP- or GDP-bound form.^5,6 PRA contains two extensive hydrophobic domains which may form a membrane-spanning domain or the inner hydrophobic core of the protein.⁷ PRA1 is localized to the Golgi and late endosomes,⁸ and the related PRA2 is present in the endoplasmic reticulum.⁹Recently it has been shown that the human YIP3 stimulates the rate of nucleotide binding to Rab9 when added to prenyl Rab9-GDI complex and catalyzes the dissociation of the endosomal Rab-GDI complex, indicating that YIP3 is a GDI displacement factor that recruits Rab to membranes.¹⁰ According to the Gougeon et al. report (2002),¹¹ PRA1 inhibits the extraction of membrane-bound Rab3A by GDI1, suggesting that recycling of Rab depends on the opposing actions of PRA and GDI, with PRA favoring membrane retention but GDI favoring solubilization.Moreover, mammalian PRA1 is required for vesicle formation from the Golgi complex and might influence the recruitment of Rab effectors during cargo sequestration as well as proteins required for subsequent vesicle docking and fusion.¹¹ This is consistent with its transport function based on interaction of yeast homologue Yip3p with proteins in the secretory pathway.⁷ Yip1-Yif1p complex binds to the ER and to the Golgi SNAREs, Bos1p and Sec22p, and is required for membrane fusion machinery.⁵ In addition, a role of Yip1p had also been proposed in COPII vesicle biogenesis.¹²To our current knowledge there is no report on the physiological role of a GDF in plants. Aims to enrich the understanding of the mechanism of Rab recycling and trafficking pathways in plant, we identified and characterized OsPRA1, a rice homolog of PRA. OsPRA1, isolated by yeast two-hybrid screening using OsRab7 as bait, localized to the prevacuolar compartment as a membrane protein.¹³ Additionally, through western blot and protoplast transient assays it was confirmed that OsPRA1 has GDF activity, which dissociates the Rab7-GDI2 complex and recruits dissociated Rab from the Rab7-GDI2 complex to the donor membrane (unpublished data). When yeast two-hybrid interaction assay between OsPRA1 and OsGDI2 was performed, OsPRA1 interacted with OsGDI2 weakly (unpublished data), supporting our proposition that OsPRA1 dissociates the OsRab7-OsGDI2 complex.Furthermore, by using yeast two-hybrid and co-immunoprecipitation assays it was demonstrated that OsPRA1 interacted with dominant negative OsRab7 (T22N) which has no GTP binding activity, but not the constitutively active OsRab7 (Q67L),¹³ indicating that OsPRA1 may interact with GDP-bound OsRab7 at the donor membrane, PVC. These results support that OsPRA1 is a GDF for OsRab7.Subsequently, in order to find its interacting proteins implicated in vesicular trafficking, such as t- or v-SNAREs, yeast two-hybrid screening using OsPRA1 as bait was performed. Interestingly, a t-SNARE, OsVam3p, homolgous to AtVam3p involved in vacuolar trafficking and localizing to both PVC and vacuole membranes in Arabidopsis,¹⁴ was isolated (unpublished data). This suggests that OsPRA1 may be a component of the vesicle fusion machinery. To further strengthen our hypothesis, we examined whether or not mutant OsPRA1 (Y94A) and OsRab7 interact. Mutant OsPRA1 (Y94A) showed weak and no interaction with OsRab7 and OsVam3p, respectively, indicating that mutant OsPRA1 (Y94A) may lose its activity for recruiting Rab GTPase and Rab effector proteins and fusing vesicles to the vacuolar membrane. Actually, when OsPRA1 was mutated, its GDF activity was reduced to less than 50%, and its localization was changed from the PVC to the cytosol. These results are consistent with the assigned transport function of OsPRA1. Besides, our data from transient expression assay using vacuole markers suggested a direct involvement of OsPRA1 in the trafficking of vacuolar proteins.In summary, OsPRA1, a Yip homologous protein, may function in regulating vacuolar trafficking as a GDF dissociating OsRab7-OsGDI2 complex in plant cells.     

Plant endosomal trafficking pathways

Endosomes regulate both the recycling and degradation of plasma membrane (PM) proteins, thereby modulating many cellular responses triggered at the cell surface. Endosomes also play a role in the biosynthetic pathway by taking proteins to the vacuole and recycling vacuolar cargo receptors. In plants, the trans-Golgi network (TGN) acts as an early/recycling endosome whereas prevacuolar compartments/multivesicular bodies (MVBs) take PM proteins to the vacuole for degradation. Recent studies have demonstrated that some of the molecular complexes that mediate endosomal trafficking, such as the retromer, the ADP-ribosylation factor (ARF) machinery, and the Endosomal Sorting Complexes Required for Transport (ESCRTs) have both conserved and specialized functions in plants. Whereas there is disagreement on the subcellular localization of the plant retromer, its function in recycling vacuolar sorting receptors (VSRs) and modulating the trafficking of PM proteins has been well established. Studies on Arabidopsis ESCRT components highlight the essential role of this complex in cytokinesis, plant development, and vacuolar organization. In addition, post-translational modifications of plant PM proteins, such as phosphorylation and ubiquitination, have been demonstrated to act as sorting signals for endosomal trafficking.     

TBC domain family, member 15 is a novel mammalian Rab GTPase-activating protein with substrate preference for Rab7

Ypt/Rabs are Ras-related GTPases that function as key regulators of intracellular vesicular trafficking. Their slow intrinsic rates of GTP hydrolysis are catalyzed by GTPase-activating proteins (GAPs). Ypt/Rab-GAPs constitute a family of proteins that contain a TBC (Tre-2/Bub2/Cdc16) domain. Only three of the 51 family members predicted in the human genome are confirmed Ypt/Rab-GAPs. Here, we report the identification and characterization of a novel mammalian Ypt/Rab-GAP, TBC domain family, member 15 (TBC1D15). TBC1D15 is ubiquitously expressed and localized predominantly to the cytosol. The TBC domain of TBC1D15 exhibits relatively high homology with that of Gyp7p, a yeast Ypt/Rab-GAP. Furthermore, TBC1D15 stimulates the intrinsic GTPase activity of Rab7, and to a lesser extent Rab11, but is essentially inactive towards Rab4 or Rab6. These data increase the number of mammalian TBC domain family members with demonstrated Rab-GAP activity to four, and suggest that TBC1D15 may be involved in Rab7-mediated late endosomal trafficking.     

Cross-talk between clathrin-dependent post-Golgi trafficking and clathrin-mediated endocytosis in Arabidopsis root cells

Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.     

RUTBC1 protein, a Rab9A effector that activates GTP hydrolysis by Rab32 and Rab33B proteins

Rab GTPases regulate all steps of membrane trafficking. Their interconversion between active, GTP-bound states and inactive, GDP-bound states is regulated by guanine nucleotide exchange factors and GTPase-activating proteins. The substrates for most Rab GTPase-activating proteins (GAPs) are unknown. Rab9A and its effectors regulate transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. We show here that RUTBC1 is a Tre2/Bub2/Cdc16 domain-containing protein that binds to Rab9A-GTP both in vitro and in cultured cells, but is not a GTPase-activating protein for Rab9A. Biochemical screening of RUTBC1 Rab protein substrates revealed highest in vitro GTP hydrolysis-activating activity with Rab32 and Rab33B. Catalysis required Arg-803 of RUTBC1, and RUTBC1 could activate a catalytically inhibited Rab33B mutant (Q92A), in support of a dual finger mechanism for RUTBC1 action. Rab9A binding did not influence GAP activity of bead-bound RUTBC1 protein. In cells and cell extracts, RUTBC1 influenced the ability of Rab32 to bind its effector protein, Varp, consistent with a physiological role for RUTBC1 in regulating Rab32. In contrast, binding of Rab33B to its effector protein, Atg16L1, was not influenced by RUTBC1 in cells or extracts. The identification of a protein that binds Rab9A and inactivates Rab32 supports a model in which Rab9A and Rab32 act in adjacent pathways at the boundary between late endosomes and the biogenesis of lysosome-related organelles.     

Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25

Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.