Expression in a RabGAP yeast mutant of two human homologues, one of which is an oncogene

The yeast proteins Msb3p and Msb4p are two Ypt/Rab-specific GTPase-activating proteins (GAPs) involved in cell growth polarization. Both proteins share with a wide variety of other proteins the highly conserved TBC domain forming the catalytically active RabGAP domain. In particular, Msb3p and Msb4p are similar to the human proteins oncTre210p (the 786-amino-acid product of the human Tre2 oncogene, implicated in Ewing's sarcoma) and RN-tre (a Rab5-GAP controlling endocytosis of the EGFR). To further understand the biochemical function of Tre2 oncogene, we expressed its cDNA and, as a control, the RN-tre cDNA, in an msb3 msb4 double mutant yeast strain. Complementation data show that RN-tre can, unlike Tre2, replace the function of the MSB3 and MSB4 genes. As two highly conserved amino acids, including the catalytic arginine, are mutated in the oncTre210p TBC domain, we restored these two amino acids and expressed the modified Tre2 cDNA in the yeast mutant.     

Mutational analysis of the <Emphasis Type="Italic">TRE2</Emphasis> oncogene encoding an inactive RabGAP

The TRE2 oncoprotein is structurally related to the RabGAP (GTPase-activating protein) family. However, TRE2 seems enzymatically inactive. Two regions are important for its lack of GAP activity. First, the TBC domain, forming the catalytically active domain of RabGAPs, is non-functional in the oncoprotein. Also involved in TRE2 inactivity is the 93-aa region flanking the TBC domain on the C-terminal side. In order to identify the residues responsible for non-functionality, we performed hydrophobic cluster analysis of the oncoprotein sequence, combined with secondary structure prediction, receptor-binding domain analysis, and a tilted peptide calculation. These analyses were complemented with site-directed and random mutagenesis experiments. On the basis of our data, we hypothesize that the lack of secondary structure of the region flanking the TBC domain in TRE2 may explain why this region plays a role in the lack of GAP activity, even when a potentially functional TBC domain is present.     

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.     

Msb4p, a protein involved in Cdc42p-dependent organization of the actin cytoskeleton, is a Ypt/Rab-specific GAP

Ypt/Rab proteins of the Ras superfamily are regulators of protein transport in exo- and endocytosis. Like Ras and Rho proteins, they have a slow intrinsic GTPase activity that can be accelerated by several orders of magnitude by GTPase-activating proteins (GAP). Here we describe a new member of a family of Ypt/Rab-specific GAPs, Msb4p/Gyp4p, that shares with other Gyp family members significant homology in the catalytic domain, recently identified in Gyp1p and Gyp7p. Purified Msb4p/Gyp4p acts primarily on Sec4p, Ypt6p and Ypt7p and might have a role in polarized secretion.     

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.     

Crystal structure of TBC1D15 GTPase‐activating protein (GAP) domain and its activity on Rab GTPases

TBC1D15 belongs to the TBC (Tre‐2/Bub2/Cdc16) domain family and functions as a GTPase‐activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark‐TBC1D15 and Sus‐TBC1D15 belong to the same subfamily of TBC domain‐containing proteins, and their GAP‐domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost.     

TBC proteins: GAPs for mammalian small GTPase Rab?

The TBC (Tre-2/Bub2/Cdc16) domain was originally identified as a conserved domain among the tre-2 oncogene product and the yeast cell cycle regulators Bub2 and Cdc16, and it is now widely recognized as a conserved protein motif that consists of approx. 200 amino acids in all eukaryotes. Since the TBC domain of yeast Gyps [GAP (GTPase-activating protein) for Ypt proteins] has been shown to function as a GAP domain for small GTPase Ypt/Rab, TBC domain-containing proteins (TBC proteins) in other species are also expected to function as a certain Rab-GAP. More than 40 different TBC proteins are present in humans and mice, and recent accumulating evidence has indicated that certain mammalian TBC proteins actually function as a specific Rab-GAP. Some mammalian TBC proteins {e.g. TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1] and TBC1D4/AS160 (Akt substrate of 160 kDa)} play an important role in homoeostasis in mammals, and defects in them are directly associated with mouse and human diseases (e.g. leanness in mice and insulin resistance in humans). The present study reviews the structure and function of mammalian TBC proteins, especially in relation to Rab small GTPases.     

Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.     

The Msb3/Gyp3 GAP controls the activity of the Rab GTPases Vps21 and Ypt7 at endosomes and vacuoles

Fusion of organelles in the endomembrane system depends on Rab GTPases that interact with tethering factors before lipid bilayer mixing. In yeast, the Rab5 GTPase Vps21 controls fusion and membrane dynamics between early and late endosomes. Here we identify Msb3/Gyp3 as a specific Vps21 GTPase-activating protein (GAP). Loss of Msb3 results in an accumulation of Vps21 and one of its effectors Vps8, a subunit of the CORVET complex, at the vacuole membrane in vivo. In agreement, Msb3 forms a specific transition complex with Vps21, has the highest activity of all recombinant GAPs for Vps21 in vitro, and is found at vacuoles despite its predominant localization to bud tips and bud necks at the plasma membrane. Surprisingly, Msb3 also inhibits vacuole fusion, which can be rescued by the Ypt7 GDP-GTP exchange factor (GEF), the Mon1-Ccz1 complex. Consistently, msb3 vacuoles fuse more efficiently than wild-type vacuoles in vitro, suggesting that GAP can also act on Ypt7. Our data indicate that GAPs such as Msb3 can act on multiple substrates in vivo at both ends of a trafficking pathway. This ensures specificity of the subsequent GEF-mediated activation of the Rab that initiates the next transport event.     

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.     

Multicopy Suppression Screen in the <Emphasis Type="Italic">msb3 msb4 Saccharomyces cerevisiae</Emphasis> Double Mutant,Affected in Ypt/RabGAP Activity

The Msb3p and Msb4p proteins of Saccharomyces cerevisiae are members of the Ypt/Rab-specific GTPase-activating protein (GAP) family. They are essential to vesicular trafficking and involved in the regulation of exocytosis and in the organization of the actin cytoskeleton, but their exact biological roles have yet to be determined. The msb3 msb4 yeast double mutation causes growth inhibition in the presence of DMSO and/or caffeine, affects the organization of the actin cytoskeleton, produces a random budding pattern in diploid cells, and affects segregation of the nucleus. To find cell components that interact genetically with the products of the MSB3 and MSB4 genes, we screened a genomic library for multicopy suppressor genes restoring normal growth of the double mutant in the presence of DMSO and caffeine. Six genes were identified, and the extent to which each gene corrects specific growth defects of the msb3 msb4 mutant is described. The encoded suppressors were classified on the basis of functional features into four groups: vesicular transport proteins (Sec7p, Vps35p, and Uso1p), a protein involved in cell division (Sap155p), a molecular chaperon (Ssz1p), and a protein associated with the 25S proteasome (Cic1p).     

Comprehensive Screening for Novel Rab‐Binding Proteins by GST Pull‐Down Assay Using 60 Different Mammalian Rabs‡

The Rab family belongs to the Ras‐like small GTPase superfamily and is implicated in membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs are yet to be identified. In this study, we systematically screened five different cell or tissue lysates for novel Rab effectors by a combination of glutathione S‐transferase (GST) pull‐down assay with 60 different mammalian Rabs and mass spectroscopic analysis. Three of the 21 Rab‐binding proteins we identified, mKIAA1055/TBC1D2B (Rab22‐binding protein), GAPCenA/TBC1D11 (Rab36‐binding protein) and centaurin β2/ACAP2 (Rab35‐binding protein), are GTPase‐activating proteins (GAPs) for Rab or Arf. Although it has recently been proposed that the Rab–GAP (Tre‐2 /Bub2/Cdc16) domain physically interacts with its substrate Rab, these three GAPs interacted with specific Rabs via a domain other than a GAP domain, e.g. centaurin β2 binds GTP‐Rab35 via the ankyrin repeat (ANKR) domain. Although centaurin β2 did not exhibit any Rab35–GAP activity in vitro, the Rab35‐binding ANKR domain of centaurin β2 was found to be required for its plasma membrane localization and regulation of Rab35‐dependent neurite outgrowth of PC12 cells through inactivation of Arf6. These findings suggest a novel mode of interaction between Rab and GAP.     

The Tre2 Oncoprotein, Implicated in Ewing’s Sarcoma, Interacts with Two Components of the Cytoskeleton

The product of the Tre2 oncogene, structurally related to the Ypt/RabGTPase-activating proteins (Ypt/RabGAP), is involved in various human cancers, including Ewing’s sarcoma. In order to identify proteins interacting with the GAP part of this protein, we performed yeast two-hybrid screening of two libraries. Two components of the cytoskeleton were thus identified, whose interaction with the GAP region was confirmed by GST-pulldown, co-immunoprecipitation, and colocalisation experiments. The proteins found to interact with the GAP region are the light regulatory chain of myosin II (Myl2) and LOC91256, a protein containing ankyrin repeats.     

Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins.

The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation.     

Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins

We present the 1.9 A resolution crystal structure of the catalytic domain of Gyp1p, a specific GTPase activating protein (GAP) for Ypt proteins, the yeast homologues of Rab proteins, which are involved in vesicular transport. Gyp1p is a member of a large family of eukaryotic proteins with shared sequence motifs. Previously, no structural information was available for any member of this class of proteins. The GAP domain of Gyp1p was found to be fully alpha-helical. However, the observed fold does not superimpose with other alpha-helical GAPs (e.g. Ras- and Cdc42/Rho-GAP). The conserved and catalytically crucial arginine residue, identified by mutational analysis, is in a comparable position to the arginine finger in the Ras- and Cdc42-GAPs, suggesting that Gyp1p utilizes an arginine finger in the GAP reaction, in analogy to Ras- and Cdc42-GAPs. A model for the interaction between Gyp1p and the Ypt protein satisfying biochemical data is given.     

Identification of Regulators for Ypt1 GTPase Nucleotide Cycling

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7 and GYP1, nor is it influenced by mutations in SEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.     

The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization

Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane. Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro. We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo. Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80-100 nm in diameter. Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants. Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane.     

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.     

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB_313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF₃ support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF₃ through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.