Arabidopsis TRAPPII is functionally linked to Rab-A,but not Rab-D in polar protein trafficking in trans-Golgi network

The trans-Golgi network (TGN) in plant cells is an independent organelle, displaying rapid association and dissociation with Golgi bodies. In plant cells, the TGN is the site where secretory and endocytic membrane trafficking meet. Cell wall components, signaling molecules and auxin transporters have been found to undergo intracellular trafficking around the TGN. However, how different trafficking pathways are regulated and how different cargoes are sorted in the TGN is poorly defined in plant cells. Using a combined approach of genetic and in vivo imaging, we recently demonstrated that Arabidopsis TRAPPII acts in the TGN and is required for polar targeting of PIN2, but not PIN1, auxin efflux carrier in root tip cells. Here, we report that, TRAPPII in Arabidopsis is required for polar distribution of AUX1, an auxin influx carrier in protophloem cells and epidermal cells of Arabidopsis root tips. In yeast cells, TRAPPII serves as a guanine-nucleotide exchange factor (GEF) for Ypt1 and Ypt31/32 in late Golgi trafficking, while in mammalian cells, TRAPPII acts as a GEF for Rab1 (homolog of yeast Ypt1) in early Golgi trafficking. We show here that TRAPPII in Arabidopsis is functionally linked to Rab-A proteins, homologs of yeast Ypt31/32, but not Rab-D proteins, homologs of yeast Ypt1 and animal Rab1 proteins.     

Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p

A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Delta strain displays a growth defect on synthetic medium at 37 degrees C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Delta cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Delta cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.     

Conservation of the TRAPPII-specific subunits of a Ypt/Rab exchanger complex

Background  

Ypt/Rab GTPases and their GEF activators regulate intra-cellular trafficking in all eukaryotic cells. In S. cerivisiae, the modular TRAPP complex acts as a GEF for the Golgi gatekeepers: Ypt1 and the functional pair Ypt31/32. While TRAPPI, which acts in early Golgi, is conserved from fungi to animals, not much is known about TRAPPII, which acts in late Golgi and consists of TRAPPI plus three additional subunits.     

Synthetic genetic array analysis of the PtdIns 4-kinase Pik1p identifies components in a Golgi-specific Ypt31/rab-GTPase signaling pathway

Phosphorylated derivatives of phosphatidylinositol are essential regulators of both endocytic and exocytic trafficking in eukaryotic cells. In Saccharomyces cerevisiae, the phosphatidylinositol 4-kinase, Pik1p generates a distinct pool of PtdIns(4)P that is required for normal Golgi structure and secretory function. Here, we utilize a synthetic genetic array analysis of a conditional pik1 mutant to identify candidate components of the Pik1p/PtdIns(4)P signaling pathway at the Golgi. Our data suggest a mechanistic involvement for Pik1p with a specific subset of Golgi-associated proteins, including the Ypt31p rab-GTPase and the TRAPPII protein complex, to regulate protein trafficking through the secretory pathway. We further demonstrate that TRAPPII specifically functions in a Ypt31p-dependent pathway and identify Gyp2p as the first biologically relevant GTPase activating protein for Ypt31p. We propose that multiple stage-specific signals, which may include Pik1p/PtdIns(4)P, TRAPPII and Gyp2p, impinge upon Ypt31 signaling to regulate Golgi secretory function.     

Two new members of a family of Ypt/Rab GTPase activating proteins. Promiscuity of substrate recognition

Monomeric GTPases of the Ras superfamily have a very slow intrinsic GTPase activity which is accelerated by specific GTPase-activating proteins. In contrast to Ras- and Rho-specific GTPase-activating proteins (GAPs) that have been studied in great detail, little is known about the functioning of GAPs specific for Ypt/Rab transport GTPases. We have identified two novel Ypt/Rab-GAPs because of their sequence relatedness to the three known GAPs Gyp1p, Gyp6p, and Gyp7p. Mdr1/Gyp2p is an efficient GAP for Ypt6p and Sec4p, whereas Msb3/Gyp3p is a potent GAP for Sec4p, Ypt6p, Ypt51p, Ypt31/Ypt32p, and Ypt1p. Although the affinity of Msb3/Gyp3p for its preferred substrate Sec4p is low (K(m) = 154 microM), it accelerates the intrinsic GTPase activity of Sec4p 5 x 10(5)-fold. Msb3/Gyp3p appears to be functionally linked to Cdc42p-regulated pathway(s). The results demonstrate that in yeast there is a large family of Ypt/Rab-GAPs, members of which discriminate poorly between GTPases involved in regulating different steps of exo- and endocytic transport routes.     

Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast

Endosomal Na+/H+ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking. We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC). The gyp6 null mutant exhibits novel phenotypes consistent with loss of negative regulation of Nhx1, including increased tolerance to hygromycin, increased vacuolar pH, and decreased plasma membrane potential. In contrast, overexpression of Gyp6 increases sensitivity to hygromycin, decreases vacuolar pH, and results in a slight missorting of vacuolar carboxypeptidase Y to the cell surface. We conclude that Gyp6 is a negative regulator of Nhx1-dependent trafficking out of the PVC. Taken together with its GTPase-activating protein-dependent role as a negative regulator of Ypt6-mediated retrograde traffic to the Golgi, we propose that Gyp6 coordinates upstream and downstream events in the PVC to Golgi pathway. Our findings provide a possible molecular link between intraendosomal pH and regulation of vesicular trafficking.     

Primary structure and biochemical characterization of yeast GTPase-activating proteins with substrate preference for the transport GTPase Ypt7p.

Small GTPases of the Ypt/Rab family are regulators of vesicular protein trafficking in exo-and endocytosis. GTPase-activating proteins (GAP) play an important role as down regulators of GTPases. We here report the molecular cloning of a novel GAP-encoding gene (GYP7, for GAP for Ypt7) by high expression from a Saccharomyces cerevisiae genomic library. The GYP7 gene encodes a hydrophilic protein with a molecular mass of 87 kDa. Comparison of its primary sequence with that of the three other known GAPs for transport GTPases, the yeast Gyp6 and Gyp1 proteins and the Rab3A-GAP from rat brain, shows similarity between the yeast GAPs only. Like GYP6 and GYP1, GYP7 is not essential for yeast cell viability. Gyp7p was able to most effectively accelerate the intrinsic GTPase activity of Ypt7p. It was also active, but to a lesser extent, on Ypt31p, Ypt32p and Ypt1p. Ypt6p, Sec4p and the human H-Ras protein did not serve as substrates. We also report the identification and cloning of a gene from the dimorphic yeast Yarrowia lipolytica that encodes a protein whose primary structure and biochemical activity are significantly related to those of Gyp7p from baker's yeast.     

TRAPPII subunits are required for the specificity switch of a Ypt-Rab GEF

Ypt-Rab GTPases are key regulators of the various steps of intracellular trafficking. Guanine nucleotide-exchange factors (GEFs) regulate the conversion of Ypt-Rabs to the GTP-bound state, in which they interact with effectors that mediate all the known aspects of vesicular transport. An interesting possibility is that Ypt-Rabs coordinate separate steps of the transport pathways. The conserved modular complex TRAPP is a GEF for the Golgi gatekeepers Ypt1 and Ypt31/32 (Refs 5-7). However, it is not known how Golgi entry and exit are coordinated. TRAPP comes in two configurations: the seven-subunit TRAPPI is required for endoplasmic reticulum-to-Golgi transport, whereas the ten-subunit TRAPPII functions in late Golgi. The two essential TRAPPII-specific subunits Trs120 and Trs130 have been identified as Ypt31/32 genetic interactors. Here, we show that they are required for switching the GEF specificity of TRAPP from Ypt1 to Ypt31. Moreover, a trs130ts mutation confers opposite effects on the intracellular localization of these GTPases. We suggest that the Trs120-Trs130 subcomplex joins TRAPP in the late Golgi to switch its GEF activity from Ypt1 to Ypt31/32. Such a 'switchable' GEF could ensure sequential activation of these Ypts, thereby coordinating Golgi entry and exit.     

Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases.

Ypt/Rab proteins constitute the largest subfamily of the Ras superfamily of monomeric GTPases and are regulators of vesicular protein transport. Their slow intrinsic GTPase activity (10(-4)-10(-3) min(-1) at 30 degrees C) has to be accelerated to switch the active to the inactive conformation. We have identified the catalytic domain within the C-terminal halves of two yeast GTPase-activating proteins (GAPs), Gyp1p and Gyp7p, with specificity for Ypt/Rab GTPases. The catalytically active fragments of Gyp1p and Gyp7p were more active than the full-length proteins and accelerated the intrinsic GTP hydrolysis rates of their preferred substrates by factors of 4.5 x 10(4) and 7.8 x 10(5), respectively. The K(m) values for the Gyp1p and Gyp7p active fragments (143 and 42 microM, respectively) indicate that the affinities of those GAPs for their substrates are very low. The catalytic domains of Gyp1p and Gyp7p contain five invariant arginine residues; substitutions of only one of them (R343 in Gyp1p and R458 in the analogous position of Gyp7p) rendered the GAPs almost completely inactive. We suggest that Ypt/Rab-GAPs, like Ras- and Rho-GAPs, follow the same mode of action and provide a catalytic arginine ('arginine finger') in trans to accelerate the GTP hydrolysis rate of the transport GTPases.     

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.     

Ypt31/32 GTPases and their novel F-box effector protein Rcy1 regulate protein recycling

Ypt/Rab GTPases control various aspects of vesicle formation and targeting via their diverse effectors. We report a new role for these GTPases in protein recycling through a novel effector. The F-box protein Rcy1, which mediates plasma membrane recycling, is identified here as a downstream effector of the Ypt31/32 GTPase pair because it binds active GTP-bound Ypt31/32 and colocalizes with these GTPases on late Golgi and endosomes. Furthermore, Ypt31/32 regulates the polarized localization and half-life of Rcy1. This suggests that Ypt/Rabs can regulate the protein level of their effectors, in addition to the established ways by which they control their effectors. We show that like Rcy1, Ypt31/32 regulate the coupled phosphorylation and recycling of the plasma membrane v-SNARE Snc1. Moreover, Ypt31/32 and Rcy1 regulate the recycling of the furin-homolog Kex2 to the Golgi. Therefore, Ypt31/32 and Rcy1 mediate endosome-to-Golgi transport, because this is the only step shared by Snc1 and Kex2. Finally, we show that Rcy1 physically interacts with Snc1. Based on this result and because F-box proteins serve as adaptors between specific substrates and ubiquitin ligases, we propose that Ypt31/32 GTPases regulate the function of Rcy1 in the phosphorylation and/or ubiquitination of proteins that recycle through the Golgi.     

A Ypt32p exchange factor is a putative effector of Ypt1p

Ypt1p regulates vesicle tethering and fusion events from the ER to the Golgi and through the early Golgi. Genetic studies have suggested a functional relationship between Ypt1p and Ypt31p/Ypt32p. Ypt31p and Ypt32p are a pair of functionally redundant GTPases that act after Ypt1p to mediate intra-Golgi traffic or the budding of post-Golgi vesicles from the trans-Golgi. Here we report that a novel Ypt32p exchange factor is a putative effector of Ypt1p. These findings implicate small GTP-binding proteins of the Ypt/Rab family in a signal cascade that directs membrane traffic through the secretory pathway.     

Specific binding to a novel and essential Golgi membrane protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p.

The regulation of vesicular transport in eukaryotic cells involves Ras-like GTPases of the Ypt/Rab family. Studies in yeast and mammalian cells indicate that individual family members act in vesicle docking/fusion to specific target membranes. Using the two-hybrid system, we have now identified a 248 amino acid, integral membrane protein, termed Yip1, that specifically binds to the transport GTPases Ypt1p and Ypt31p. Evidence for physical interaction of these GTPases with Yip1p was also demonstrated by affinity chromatography and/or co-immunoprecipitation. Like the two GTPases, Yip1p is essential for yeast cell viability and, according to subcellular fractionation and indirect immunofluorescence, is located to Golgi membranes at steady state. Mutant cells depleted of Yip1p and conditionally lethal yip1 mutants at the non-permissive temperature massively accumulate endoplasmic reticulum membranes and display aberrations in protein secretion and glycosylation of secreted invertase. The results suggests for a role for Yip1p in recruiting the two GTPases to Golgi target membranes in preparation for fusion.     

Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase

Rab guanosine triphosphatases (GTPases) are pivotal regulators of membrane identity and dynamics, but the in vivo pathways that control Rab signaling are poorly defined. Here, we show that the GTPase-activating protein Gyp7 inactivates the yeast vacuole Rab Ypt7 in vivo. To efficiently terminate Ypt7 signaling, Gyp7 requires downstream assistance from an inhibitory casein kinase I, Yck3. Yck3 mediates phosphorylation of at least two Ypt7 signaling targets: a tether, the Vps-C/homotypic fusion and vacuole protein sorting (HOPS) subunit Vps41, and a SNARE, Vam3. Phosphorylation of both substrates is opposed by Ypt7-guanosine triphosphate (GTP). We further demonstrate that Ypt7 binds not one but two Vps-C/HOPS subunits: Vps39, a putative Ypt7 nucleotide exchange factor, and Vps41. Gyp7-stimulated GTP hydrolysis on Ypt7 therefore appears to trigger both passive termination of Ypt7 signaling and active kinase-mediated inhibition of Ypt7's downstream targets. We propose that signal propagation through the Ypt7 pathway is controlled by integrated feedback and feed-forward loops. In this model, Yck3 enforces a requirement for the activated Rab in docking and fusion.     

COPI–TRAPPII activates Rab18 and regulates its lipid droplet association

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII‐specific subunits by various methods including siRNA depletion and CRISPR–Cas9‐mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII‐deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD. We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.     

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.     

Structural insights into assembly of TRAPPII and its activation of Rab11/Ypt32

Transport protein particle (TRAPP) complexes belong to the multisubunit tethering complex. They are guanine nucleotide exchange factors (GEFs) that play essential roles in secretory and endocytic recycling pathway and autophagy. There are two major forms of TRAPP complexes, TRAPPII and TRAPPIII, which share a core set of small subunits. TRAPPIII activates Rab1, while TRAPPII primarily activates Rab11. A steric gating mechanism has been proposed to control the substrate selection in vivo. However, the detailed mechanisms underlying the transition from TRAPPIII's GEF activity for Rab1 to TRAPPII's GEF activity for Rab11 and the roles of the complex-specific subunits in this transition are insufficiently understood. In this review, we discuss recent advances in understanding the mechanism of specific activation of Rab11/Ypt32 by TRAPPII, with a particular focus on new findings from structural studies.     

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4

Dysfunctions of primary cilia and cilia‐derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans‐Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase‐activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post‐TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1‐mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4‐based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.     

Two GTPase isoforms, Ypt31p and Ypt32p, are essential for Golgi function in yeast.

In eukaryotic cells, monomeric GTPases of the Ypt/Rab family function as regulators at defined steps of vesicular transport in exo- and endocytosis. Here we report on the isolation and characterization of two genes (YPT31 and YPT32) of the yeast Saccharomyces cerevisiae which encode members of the Ypt family exhibiting >80% sequence identity. Whereas the disruption of one of the two genes was phenotypically neutral, the disruption of both YPT31 and YPT32 led to lethality. Depletion of wild-type Ypt31p or of a short-lived ubiquitin-Ypt31p in a ypt32 null background led to a massive accumulation of Golgi-like membranes, an inhibition of invertase secretion and defects in vacuolar protein maturation. Similar alterations were observed in a conditional-lethal ypt31-1 mutant at 30 min after shift to the non-permissive temperature. According to subcellular fractionation, a significant part of Ypt31p appeared to be located in Golgi-enriched membrane fractions. In accordance with this, indirect immunofluorescence using affinity-purified anti-Ypt31p antibodies gave a punctate staining similar to that observed with Golgi-located proteins. From the phenotypic alterations observed in ypt31 and ypt32 mutants, it seems likely that the two GTPases are involved in intra-Golgi transport or in the formation of transport vesicles at the most distal Golgi compartment.     

Functional implications of genetic interactions between genes encoding small GTPases involved in vesicular transport in yeast

Ras-related, guanine nucleotide-binding proteins of the Ypt/Rab family play a key role at defined steps in vesicular transport, both in yeast and in mammalian cells. In yeast, Ypt1p has an essential function late in endoplasmic reticulum (ER) to Golgi transport, and the redundant Ypt31/Ypt32 GTPases have been proposed to act in transport through and/or from the Golgi. Here we report that mutant alleles of YPT31 and YPT32, whose gene products have a reduced affinity for GTP, are able to suppress the dominant lethal phenotype of YPT1 ^N121I . Co-expression of YPT1 ^N121I and the suppressor YPT31 ^N126I allow essentially undisturbed secretory transport in the absence of the respective wild-type GTPases. Such mutant cells massively overaccumulate 60–100 nm vesicles and are heat sensitive. It appears likely that the mutant GTPases, which are defective in nucleotide binding, compete for the binding of common interacting protein(s). These and other genetic interactions between YPT1, YPT31/32, ARF1 and SEC4 described here strongly support the view that Ypt31p and Ypt32p have a central, Golgi-associated function in anterograde or retrograde transport. Received: 28 August 1998 / Accepted: 14 October 1998