Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy

Membrane trafficking is regulated by small Ras-like GDP/GTP binding proteins of the Rab subfamily (Rab GTPases) that cycle between membranes and cytosol depending on their nucleotide state. The GDP dissociation inhibitor (GDI) solubilizes prenylated Rab GTPases from and shuttles them between membranes in the form of a soluble cytosolic complex. We use attenuated total reflection–Fourier transform infrared spectroscopy to directly observe extraction of Rab GTPases from model membranes by GDI. In their native form, most Rab GTPases are doubly geranylgeranylated at the C terminus to achieve localization to the membrane. We find that monogeranylgeranylated Rab35 and Rab1b reversibly bind to a negatively charged model membrane. Correct folding and GTPase activity of the membrane-bound protein can be evaluated. The dissociation kinetics depends on the C-terminal sequence and charge of the GTPases. The attenuated total reflection experiments show that GDI genuinely accelerates the intrinsic Rab membrane dissociation. The extraction process is characterized and occurs in a nucleotide-dependent manner. Furthermore, we find that phosphocholination of Rab35, which is catalyzed by the Legionella pneumophila protein AnkX, interferes with the ability of GDI to extract Rab35 from the membrane. The attenuated total reflection–Fourier transform infrared spectroscopy approach enables label-free investigation of the interaction between GDI and Rab GTPases in a membrane environment. Thereby, GDI is revealed to actively extract monogeranylgeranylated membrane-bound Rab GTPases and, thus, is not merely a solubilization factor.     

Structural mechanism of host Rab1 activation by the bifunctional Legionella type IV effector SidM/DrrA

Bacterial pathogens deliver effector proteins with diverse biochemical activities into host cells, thereby modulating various host functions. Legionella pneumophila hijacks host vesicle trafficking to avoid phagosome–lysosome fusion, a mechanism that is dependent on the Legionella Dot/Icm type IV secretion system. SidM/DrrA, a Legionella type IV effector, is important for the interactions of Legionella-containing vacuoles with host endoplasmic reticulum–derived vesicles. SidM is the only known protein that catalyzes both the exchange of GDP for GTP and GDI displacement from small GTPase Rab1. We determined the crystal structures of SidM alone (residues 317–647) and SidM (residues 193–550) in complex with nucleotide-free WT Rab1. The SidM structure contains an N-terminal helical domain with a potential new function, a Rab1-activation domain, and a C-terminal phosphatidylinositol 4-phosphate–binding P4M domain. The Rab1-activation domain has extensive strong interactions mainly with Rab1 switch I and II regions that undergo substantial conformational changes on SidM binding. Mutations of switch-contacting residues in SidM attenuate both the nucleotide exchange and GDI displacement activities. Structural comparisons of Rab1 in the SidM complex with Rab1-GDP and Ypt1-GDP in the GDI complex identify key conformational changes that disrupt the nucleotide and GDI binding of Rab1. Further biochemical and structural analyses reveal a unique mechanism of coupled GDP release and GDI displacement likely triggered by the SidM-induced drastic displacement of switch I of Rab1.     

GTPase activating protein activity for Rab4 is enriched in the plasma membrane of 3T3-L1 adipocytes. Possible involvement in the regulation of Rab4 subcellular localization

Summary The small guanosine 5-triphosphate (GTP)ase Rab4 has been suggested to play a role in insulin-induced GLUT4 translocation. Under insulin stimulation, GLUT4 translocates to the plasma membranes, while Rab4 leaves the GLUT4-containing vesicles and becomes cytosolic. Rab proteins cycle between a GTP-bound active form and a guanosine 5-diphosphate (GDP)-bound inactive form. The intrinsic GTPase activity of Rab proteins is low and the interconversion between the two forms is dependent on accessory factors. In the present work, we searched for a GTPase activating protein (GAP) for Rab4 in 3T3-L1 adipocytes. We used a glutathione-S-transferase (GST)-Rab4 protein which possesses the properties of a small GTPase (ability to bind GDP and GTP and to hydrolyse GTP) and can be isolated in a rapid and efficient way. This GAP activity was observed in 3T3-L1 adipocyte lysates, and was able to accelerate the hydrolysis of the [-³²P]GTP bound to GST-Rab4 into [-³²P]GDP. This activity, tentatively called Rab4-GAP, was also present in 3T3-L1 fibroblasts. The Rab4-GAP activity was present in total membrane fractions and nearly undetectable in cytosol. Following subcellular fractionation, Rab4-GAP was found to be enriched in plasma membranes when compared to internal microsomes. Insulin treatment of the cells had no effect on the total Rab4-GAP activity or on its subcellular localization. Taking our results together with the accepted model of Rab cycling in intracellular traffic, we propose that Rab4-GAP activity plays a role in the cycling between the GTP- and GDP-bound forms of Rab4, and thus possibly in the traffic of GLUT4-containing vesicles.Abbreviations GAP GTPase activating protein - GDI guanosine dissociation inhibitor - GDS guanosine dissociation stimulator - GDF GDI dissociation factor - GEF GDP exchange factor - GST glutathione-S-transferase - p44^mapk MAP-kinase isoform with an M_r 44000 - PM plasma membranes - HLDM high and low density microsomes - DMEM Dulbecco's modified Eagle's medium - BSA bovine serum albumin - PVDF polyvinylidene difluoride - KLH Keyhole limpet haemocyanin - CHAPS 3-[(3-cholamidopropyl)dimethylammonic]-1-propane sulphonate - AS subunit of G_i1,2     

The role of the hypervariable C-terminal domain in Rab GTPases membrane targeting

Intracellular membrane trafficking requires correct and specific localization of Rab GTPases. The hypervariable C-terminal domain (HVD) of Rabs is posttranslationally modified by isoprenyl moieties that enable membrane association. A model asserting HVD-directed targeting has been contested in previous studies, but the role of the Rab HVD and the mechanism of Rab membrane targeting remain elusive. To elucidate the function of the HVD, we have substituted this region with an unnatural polyethylenglycol (PEG) linker by using oxime ligation. The PEGylated Rab proteins undergo normal prenylation, underlining the unique ability of the Rab prenylation machinery to process the Rab family with diverse C-terminal sequences. Through localization studies and functional analyses of semisynthetic PEGylated Rab1, Rab5, Rab7, and Rab35 proteins, we demonstrate that the role of the HVD of Rabs in membrane targeting is more complex than previously understood. The HVD of Rab1 and Rab5 is dispensable for membrane targeting and appears to function simply as a linker between the GTPase domain and the membrane. The N-terminal residues of the Rab7 HVD are important for late endosomal/lysosomal localization, apparently due to their involvement in interaction with the Rab7 effector Rab-interacting lysosomal protein. The C-terminal polybasic cluster of the Rab35 HVD is essential for plasma membrane (PM) targeting, presumably because of the electrostatic interaction with negatively charged lipids on the PM. Our findings suggest that Rab membrane targeting is dictated by a complex mechanism involving GEFs, GAPs, effectors, and C-terminal interaction with membranes to varying extents, and possibly other binding partners.Rab proteins are key regulators of intracellular vesicle transport in eukaryotic cells (1, 2). They comprise the largest subgroup of the Ras superfamily of small GTPases, with more than 60 members in humans and 11 members in yeast (3). Interacting with a complex network of Rab regulators and effectors, Rab GTPases regulate these processes through a spatiotemporally controlled GTPase cycle and their distribution in cells. The GTPase cycle is strictly regulated by guanine nucleotide exchange factors (GEFs) that mediate GDP/GTP exchange and by GTPase-activating proteins (GAPs) that accelerate the hydrolysis of GTP. Active (GTP-bound) Rab proteins associate with distinct intracellular compartments and direct vesicular transport by recruiting a multitude of Rab-specific effectors, including tethering complexes and motor proteins.Rab proteins are posttranslationally modified at the C terminus with prenyl groups that function as membrane anchors. Rab prenylation involves covalent attachment of the geranylgeranyl (C-20 isoprenyl) moiety to one or two C-terminal cysteine residues of the protein substrate via a stable thioether linkage (4). Unlike other protein prenyltransferases that recognize the C-terminal CaaX motif of protein substrates (e.g., Ras and Rho), Rab geranylgeranyl transferase (RabGGTase) does not recognize its protein substrates (Rab proteins) directly but requires the adaptor Rab escort protein (REP). Rab prenylation requires the formation of a ternary catalytic Rab:REP:RabGGTase complex (5–7). It remains elusive how the single Rab prenylation machinery can process the whole Rab family with diverse C termini.Cycling between the cytosol and membranes is an essential feature of the mode of action of Rabs and is made possible by reversible interaction with GDP dissociation inhibitor (GDI), which can solubilize the otherwise water-insoluble geranylgeranylated Rab molecules (8). Membrane-bound GDI displacement factors were proposed to disrupt GDI:Rab complexes, leading to insertion of the prenylated Rab into the membrane in the GDP form and release of GDI into the cytosol (9, 10). One of the most perplexing questions is how Rab proteins are specifically targeted to their cognate membranes. The hypervariable C-terminal domain (HVD) was proposed to function as a signal for targeting Rab proteins to specific subcellular membranes (11). However, later studies suggested that several features of Rab molecules, Rab effectors, and GEFs are involved in the targeting process (12–15). The role of the Rab HVD in membrane targeting is controversial, because contradictory results were obtained by swapping the hypervariable domains of Rab proteins (11–13). Thus, the function of the Rab HVD and a complete model for Rab membrane targeting remain to be established. To further understand the significance of the HVD in Rab membrane targeting and prenylation, unique methods are needed to manipulate the structure of Rab C terminus.In this study, we replaced the Rab C-terminal sequence with the polyethylenglycol (PEG) linker as a nonpeptidic chain. The PEG chain containing two cysteine residues or a simple thiol group at one end was coupled to truncated Rab proteins by oxime ligation. We found that the PEGylated Rab proteins undergo normal prenylation in vitro, confirming that the Rab prenylation machinery does not require a specific C-terminal sequence but rather outsources the specificity to the REP molecule. By combining this semisynthetic strategy with cell imaging, we elucidate the role of the hypervariable C-terminal domain for subcellular Rab targeting. In some instances, the HVD is dispensable for correct subcellular localization (Rab1, Rab5), but is essential in other cases because of specific interactions with effectors (Rab7) or electrostatic interactions with membranes (Rab35). The results further elaborate the model for Rab prenylation and membrane targeting.     

Regulation of phagocyte function by low molecular weight GTP-binding proteins

Abstract: The mechanisms used by phagocytic leukocytes in the process of bacterial killing are regulated by GTP-binding proteins of the Ras superfamily. In particular, the formation of toxic oxygen metabolites via the NADPH oxidase requires the action of both Rac and Rap1A proteins. Rac2 forms a third cytosolic component of the human neutrophil NADPH oxidase. Rac2 is active in its GTP-bound form, and requires post-translational processing (isoprenylation) in order to interact with regulatory proteins which stimulate the exchange of GTP for GDP. In the resting neutrophil, Rac is localized to the cytosol in the form of a complex with a GDP dissociation inhibitor (GDI) protein. Upon cell activation, this complex is disrupted to enable Rac to translocate to the active oxidase at the plasma membrane. The Rac-GDI complex may be regulated by the release of specific lipids known to be generated during phagocyte activation.     

Rab4 facilitates cyclic adenosine monophosphate-stimulated bile acid uptake and Na+-taurocholate cotransporting polypeptide translocation

Cyclic adenosine monophosphate (cAMP) stimulates hepatic bile acid uptake by translocating sodium-taurocholate (TC) cotransporting polypeptide (Ntcp) from an endosomal compartment to the plasma membrane. Rab4 is associated with early endosomes and involved in vesicular trafficking. This study was designed to determine the role of Rab4 in cAMP-induced TC uptake and Ntcp translocation. HuH-Ntcp cells transiently transfected with empty vector, guanosine triphosphate (GTP) locked dominant active Rab4 (Rab4(GTP)), or guanosine diphosphate (GDP) locked dominant inactive Rab4 (Rab4(GDP)) were used to study the role of Rab4. Neither Rab4(GTP) nor Rab4(GDP) affected either basal TC uptake or plasma membrane Ntcp level. However, cAMP-induced increases in TC uptake and Ntcp translocation were enhanced by Rab4(GTP) and inhibited by Rab4(GDP). In addition, cAMP increased GTP binding to endogenous Rab4 in a time-dependent, but phosphoinositide-3-kinase-independent manner. CONCLUSION: Taken together, these results suggest that cAMP-mediated phosphoinositide-3-kinase-independent activation of Rab4 facilitates Ntcp translocation in HuH-Ntcp cells.     

Interaction analysis of prenylated Rab GTPase with Rab escort protein and GDP dissociation inhibitor explains the need for both regulators

Prenylated Rab GTPases regulate intracellular vesicle trafficking in eukaryotic cells by associating with specific membranes and recruiting a multitude of Rab-specific effector proteins. Prenylation, membrane delivery, and recycling of all 60 members of the Rab GTPase family are regulated by two related molecules, Rab escort protein (REP) and GDP dissociation inhibitor (GDI). Biophysical analysis of the interaction of prenylated proteins is complicated by their low solubility in aqueous solutions. Here, we used expressed protein ligation to construct a semisynthetic fluorescent analogue of prenylated Rab7, Rab7-NBD-farnesyl. This molecule is soluble in the absence of detergent but is otherwise similar in its behavior to naturally prenylated Rab7 GTPase. To obtain information on the interaction of natively mono- and diprenylated Rab7 GTPases with REP and GDI molecules, we stabilized the former molecules in solution by using the beta-subunit of Rab geranylgeranyl transferase, which we demonstrate to function as an unspecific chaperone of prenylated proteins. Using competitive titrations of mixtures of natively prenylated and fluorescent Rab, we demonstrate that monogeranylgeranylated Rab7 binds to the REP protein with a K(d) value of approximately 70 pM. The affinity of doubly prenylated Rab7 is approximately 20-fold weaker. In contrast, GDI binds both prenylated forms of Rab7 with comparable affinities (K(d) = 1-5 nM) but has extremely low affinity to unprenylated Rab molecules. The obtained data allow us to formulate a thermodynamic model for the interaction of RabGTPases with their regulators and membranes and to explain the need for both REP and GDI in Rab function.     

Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate

Rab GTPases are key regulators of membrane traffic pathways within eukaryotic cells. They are specifically activated by guanine nucleotide exchange factors (GEFs), which convert them from their “inactive” GDP-bound form to the “active” GTP-bound form. In higher eukaryotes, proteins containing DENN-domains comprise a major GEF family. Here we describe at 2.1-Å resolution the first structure of a DENN-domain protein, DENND1B-S, complexed with its substrate Rab35, providing novel insights as to how DENN-domain GEFs interact with and activate Rabs. DENND1B-S is bi-lobed, and interactions with Rab35 are through conserved surfaces in both lobes. Rab35 binds via switch regions I and II, around the nucleotide-binding pocket. Positional shifts in Rab residues required for nucleotide binding may lower its affinity for bound GDP, and a conformational change in switch I, which makes the nucleotide-binding pocket more solvent accessible, likely also facilitates exchange.In eukaryotic cells, material is transported between membrane-bound organelles or the plasma membrane by vesicles. These bud from the membrane of the donor organelle, travel to and are recognized at the target compartment, and finally fuse with it to deliver their cargo. Vesicle traffic is orchestrated by a large number of proteins, including coat proteins that facilitate budding, tethering complexes involved in vesicle recognition, SNARE proteins that drive membrane fusion, and small GTPases in the Rab/Ypt family that have regulatory roles (1). Different subsets of these proteins are involved in different transport pathways.Rab GTPases are key determinants of organelle identity and hence in ensuring that vesicular cargo is delivered to the correct destination (2–4). The Rabs themselves are activated at the appropriate membranes by Rab-specific guanine nucleotide exchange factors (GEFs), which facilitate the conversion of the Rab from its inactive, GDP-bound, to the active, GTP-bound state. The GEF interacts with its Rab substrate to lower its nucleotide-binding affinity, accelerating the departure of bound GDP and allowing GTP, which is 10-fold more abundant in the cell than GDP, to bind (5, 6). In their GTP-bound membrane associated form, Rabs then recruit additional proteins to mediate vesicle recognition, tethering, and fusion events.Mammals have more than 60 different Rabs (7), with GEFs that activate most of these yet to be identified. Recent data suggest that a large subset of mammalian Rabs, or at least 10 different Rab GTPases, are activated by proteins containing a DENN domain (8). In humans, eighteen different DENN-domain proteins have been identified, including members of the DENND1 through DENND5 families, the myotubularin-related proteins MTMR5 and 13, and the MAP-kinase activating death domain MADD (8, 9). Studies of the DENND1 family have shown that the DENN domain itself is sufficient for GEF activity (10, 11), where both DENND1A and 1B activate Rab35 for its role in the endocytic pathway (8, 10, 11). The substrate specificity of DENND1C remains unclear (8), although it has also been proposed as a Rab35 GEF (8, 11).Despite their importance in regulating membrane traffic in higher eukaryotes, little is known regarding the structure of DENN domains or the mechanisms by which they function. Here, to obtain first insights regarding how these proteins recognize and activate their Rab partners, we have determined at 2.1-Å resolution the structure of the DENN domain from DENND1B in complex with the nucleotide-free form of its substrate Rab35. The complex represents an intermediate on the reaction pathway, after GDP has left the Rab35 nucleotide-binding pocket and before GTP binding.     

Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis

Two so-called "secretory Rabs," Rab3 and Rab27, regulate late steps during dense-core vesicle exocytosis in neuroendocrine cells. Sperm contain a single large dense-core granule that is released by regulated exocytosis (termed the acrosome reaction) during fertilization or on exposure to inducers in vitro. Sperm exocytosis uses the same fusion machinery as neurons and neuroendocrine cells, with an additional requirement for active Rab3. Here we show that Rab27 is also required for the acrosome reaction, as demonstrated by the inability of inducers to elicit exocytosis when streptolysin O-permeabilized human sperm were loaded with inhibitory anti-Rab27 antibodies or the Rab27-GTP binding domain of the effector Slac2-b. The levels of GTP-bound Rab27 increased on initiation of exocytosis, as did the proportion of GTP-bound Rab3A. We have developed a fluorescence microscopy-based method for detecting endogenous Rab3A-GTP and Rab27-GTP in the acrosomal region of human sperm. Challenge with an inducer increased the population of cells exhibiting GTP-bound Rabs in this subcellular domain. Interestingly, introducing recombinant Rab27A loaded with GTP-γ-S into sperm elicited a remarkable increase in the number of cells evincing GTP-bound Rab3A. In the converse condition, recombinant Rab3A did not modify the percentage of Rab27-GTP-containing cells. Furthermore, Rab27A-GTP recruited a Rab3 GDP/GTP exchange factor (GEF) activity. Our findings suggest that Rab27/Rab3A constitutes a Rab-GEF cascade in dense-core vesicle exocytosis.     

A single residue can modify target-binding affinity and activity of the functional domain of the Rho-subfamily GDP dissociation inhibitors.

The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins for the Rho- and Rab-subtype GTP-binding proteins. As a first step toward identifying the key functional domain(s) on the Rho-subtype GDI, truncations of the amino and carboxyl termini were performed. Deletion of the final four amino acids from the carboxyl terminus of Rho GDI or the removal of 25 amino acids from the amino terminus had no significant effect on the ability of the GDI to inhibit GDP dissociation from the Rho-like protein Cdc42Hs or on its ability to release Cdc42Hs from membrane bilayers. However, the deletion of 8 amino acids from the carboxyl terminus of Rho GDI eliminated both activities. To further test the importance of the carboxyl-terminal domain of the Rho GDI molecule, chimeras were constructed between this GDI and a related protein designated LD4, which is 67% identical to Rho GDI but is less potent by a factor of 10-20 than Rho GDI in functional assays with the Cdc42Hs protein. Two sets of chimeras were constructed that together indicated that as few as 6 amino acids near the carboxyl terminus of Rho GDI could impart full GDP dissociation inhibition and membrane dissociation activities on the LD4 molecule. Further analysis of this region by site-directed mutagenesis showed that a single change at residue 174 of LD4 to the corresponding residue of Rho GDI (i.e., Asp-174-->Ile) could impart nearly full (70%) Rho GDI activity on the LD4 molecule.     

Rabs and their effectors: achieving specificity in membrane traffic

Rab proteins constitute the largest branch of the Ras GTPase superfamily. Rabs use the guanine nucleotide-dependent switch mechanism common to the superfamily to regulate each of the four major steps in membrane traffic: vesicle budding, vesicle delivery, vesicle tethering, and fusion of the vesicle membrane with that of the target compartment. These different tasks are carried out by a diverse collection of effector molecules that bind to specific Rabs in their GTP-bound state. Recent advances have not only greatly extended the number of known Rab effectors, but have also begun to define the mechanisms underlying their distinct functions. By binding to the guanine nucleotide exchange proteins that activate the Rabs certain effectors act to establish positive feedback loops that help to define and maintain tightly localized domains of activated Rab proteins, which then serve to recruit other effector molecules. Additionally, Rab cascades and Rab conversions appear to confer directionality to membrane traffic and couple each stage of traffic with the next along the pathway.     

Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation

Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate–binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.

Rab GTPases are molecular switches that function as markers of organelle identity and coordinate intracellular trafficking as part of the conserved fusion machinery (1). The cycling of Rab GTPases between the inactive GDP-bound and the active GTP-bound form is tightly controlled by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) (2). While GAPs promote the intrinsically low GTP hydrolysis rate of Rabs to switch them off, GEFs stimulate nucleotide release and the loading of Rab with GTP to convert the GTPase to its active conformation. Inactive Rabs are kept cytosolic by the GDP dissociation inhibitor (GDI), which binds the Rab prenyl anchor (3). GDI can be removed from Rab GTPases by the GDI displacement factor (4), and the exchange of GDP with GTP also couples the association of Rab GTPases with membranes. Thus, the spatiotemporal regulation of Rab GTPases and downstream fusion events ultimately depend on the activation of their cognate GEFs.Tri Longin domain (TLD) Rab-GEFs comprise one of many GEF families known. They contain at least two subunits, each of which is predicted to consist of three Longin domains (LDs) (5, 6). The TLD GEF family comprises the universally conserved Rab7-GEF Mon1-Ccz1 (MC1) (7–9) and two other complexes specific to metazoans, namely BLOC-3 (biogenesis of lysosome-related organelles complex-3, which includes Hps1 and Hps4) and Inturned-Fuzzy. BLOC-3 is the GEF for Rab32 and Rab38 on lysosome-related organelles (10), and mutations in BLOC-3 cause the genetic disease Hermansky–Pudlak syndrome (11). Inturned-Fuzzy, which acts as the GEF of Rab23 (5), has been implicated in the establishment of planar cell polarity and ciliogenesis and was described as part of the planar cell polarity effector complex in flies and the CPLANE (ciliogenesis and planar cell polarity effector) complex in mammalian cells (12). Several studies showed that in metazoans, TLD RabGEFs do not consist of two subunits but have additional non-TLD proteins bound to the heterodimeric core (13–17). The function of these additional subunits in the “enlarged” TLD RabGEF complexes is currently not clear.The best-studied TLD Rab-GEF is MC1, which activates Rab7 (Ypt7 in yeast) in endosomal maturation and autophagy. In yeast, it has been demonstrated that MC1-dependent recruitment of Ypt7 to both late endosomes/multivesicular bodies and autophagosomes is required for the fusion of these organelles with the vacuole and degradation of the respective cargo (7, 18). This process is conserved in plants and mammalian cells (9, 14, 19). The metazoan MC1 complex contains a third subunit, namely Bulli/RMC1; however, the function of this protein remains elusive (14–16). Importantly, Bulli/RMC1 is not required for Rab5-dependent Rab7 activation and is thus not involved in regulating the GEF activity of MC1 (20). Recently, the uncharacterized protein C5orf51 was identified as an interactor of MC1 that links the GEF complex to mitophagy (17), yet the underlying mechanism remains to be determined.We have previously identified the structure of Ypt7 bound to the MC1 core, which comprises a heterodimeric complex of the first Longin domains of Mon1 and Ccz1, respectively (21). The structure revealed a mechanism that involves remodeling of the GTPase switch regions. In the GEF-bound conformation, MC1 binding opens the nucleotide-binding pocket of Ypt7, which directs a lysine residue of Ypt7 into the Mg²⁺-binding pocket, thus favoring displacement of the bound nucleotide.Although the MC1 core is required and sufficient for the GEF activity of the complex (21), functional studies in yeast showed that the catalytic core complex was unable to rescue the vacuolar fragmentation phenotype of mon1Δ or ccz1Δ strains and did not properly localize in cells. Thus, LD2 and LD3 as well as the rest of the complex are likely involved in correct recruitment of MC1 to the proper organelle membrane. Previous studies have identified GTPases of the Rab5 family and phosphatidylinositol phosphate (PIP) lipids as recruiting factors that promote binding of MC1 to endosomal membranes (20, 22, 23). On autophagosomes, Atg8 supports Mon1-Ccz1 function (16, 18). However, the mechanistic basis underlying these processes remains unclear.To gain molecular insight into the targeting mechanism of MC1 and to understand how the complex is built in three dimensions, we determined the cryogenic electron microscopy (cryo-EM) structure of a stable MC1 complex from Chaetomium thermophilum. We observe a unique arrangement of the three LDs of each subunit within the complex and identify a conserved basic surface on MC1 that defines the orientation of the complex on lipid bilayers. Based on this, we developed a model of the function of Mon1-Ccz1 on membranes. The structure of Mon1-Ccz1 thus provides a blueprint for the architecture and function of the TLD RabGEF family.     

Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination

Effectors delivered into host cells by the Legionella pneumophila Dot/Icm type IV transporter are essential for the biogenesis of the specialized vacuole that permits its intracellular growth. The biochemical function of most of these effectors is unknown, making it difficult to assign their roles in the establishment of successful infection. We found that several yeast genes involved in membrane trafficking, including the small GTPase Ypt1, strongly suppress the cytotoxicity of Lpg0695(AnkX), a protein known to interfere severely with host vesicle trafficking when overexpressed. Mass spectrometry analysis of Rab1 purified from a yeast strain inducibly expressing AnkX revealed that this small GTPase is modified posttranslationally at Ser(76) by a phosphorylcholine moiety. Using cytidine diphosphate-choline as the donor for phosphorylcholine, AnkX catalyzes the transfer of phosphorylcholine to Rab1 in a filamentation-induced by cAMP(Fic) domain-dependent manner. Further, we found that the activity of AnkX is regulated by the Dot/Icm substrate Lpg0696(Lem3), which functions as a dephosphorylcholinase to reverse AnkX-mediated modification on Rab1. Phosphorylcholination interfered with Rab1 activity by making it less accessible to the bacterial GTPase activation protein LepB; this interference can be alleviated fully by Lem3. Our results reveal reversible phosphorylcholination as a mechanism for balanced modulation of host cellular processes by a bacterial pathogen.     

Ly-GDI, a GDP-dissociation inhibitor of the RhoA GTP-binding protein, is expressed preferentially in lymphocytes.

The Ras-related small GTP-binding proteins are involved in diverse cellular events, including cell signaling, proliferation, cytoskeletal organization, and secretion. The interconversion of the active, GTP-bound form of the protein to the inactive, GDP-bound form is influenced by two types of regulatory proteins, those that alter the intrinsic GTPase activity of the GTP-binding protein and those that affect the rate of GDP/GTP exchange. By utilizing a subtractive hybridization approach, we have isolated a human gene encoding Ly-GDI, a protein that has striking homology to the product of a previously cloned gene, Rho-GDI, which inhibits GDP/GTP exchange on the Rho family of GTPases. In contrast to Rho-GDI, which is ubiquitously expressed, Ly-GDI is expressed only in hematopoietic tissues and predominantly in B- and T-lymphocyte cell lines. The full-length Ly-GDI cDNA encodes a 27-kDa protein which binds to RhoA and inhibits GDP dissociation from RhoA. Stimulation of T lymphocytes with phorbol ester leads to phosphorylation of Ly-GDI, suggesting an involvement of Ly-GDI in lymphocyte activation pathways. Cell type-specific regulators of the Ras-like GTP-binding proteins may provide one mechanism by which different cell types respond uniquely to signals transduced through the same cell surface receptor or may provide a way by which the GTP-binding proteins can be uniquely engaged by tissue-restricted receptors.     

Activator of G protein signaling 3 is a guanine dissociation inhibitor for Galpha i subunits

Activator of G protein signaling 3 (AGS3) is a newly identified protein shown to act at the level of the G protein itself. AGS3 belongs to the GoLoco family of proteins, sharing the 19-aa GoLoco motif that is a Galpha(i/o) binding motif. AGS3 interacts only with members of the Galpha(i/o) subfamily. By surface plasmon resonance, we found that AGS3 binds exclusively to the GDP-bound form of Galpha(i3). In GTPgammaS binding assays, AGS3 behaves as a guanine dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP by Galpha(i3). AGS3 interacts with both Galpha(i3) and Galpha(o) subunits, but has GDI activity only on Galpha(i3), not on Galpha(o). The fourth GoLoco motif of AGS3 is a major contributor to this activity. AGS3 stabilizes Galpha(i3) in its GDP-bound form, as it inhibits the increase in tryptophan fluorescence of the Galpha(i3)-GDP subunit stimulated by AlF(4)(-). AGS3 is widely expressed as it is detected by immunoblotting in brain, testis, liver, kidney, heart, pancreas, and in PC-12 cells. Several different sizes of the protein are detected. By Northern blotting, AGS3 shows 2.3-kb and 3.5-kb mRNAs in heart and brain, respectively, suggesting tissue-specific alternative splicing. Taken together, our results demonstrate that AGS3 is a GDI. To the best of our knowledge, no other GDI has been described for heterotrimeric G proteins. Inhibition of the Galpha subunit and stimulation of heterotrimeric G protein signaling, presumably by stimulating Gbetagamma, extend the possibilities for modulating signal transduction through heterotrimeric G proteins.     

Rab8A and Rab13 are activated by insulin and regulate GLUT4 translocation in muscle cells

Skeletal muscle is the primary site of dietary glucose disposal, a function that depends on insulin-mediated exocytosis of GLUT4 vesicles to its cell surface. In skeletal muscle and adipocytes, this response involves Akt signaling to the Rab-GAP (GTPase-activating protein) AS160/TBC1D4. Intriguingly, the AS160-targeted Rabs appear to differ, with Rab8A participating in GLUT4 exocytosis in muscle cells and Rab10 in adipocytes, and their activation by insulin is unknown. Rabs 8A, 10, and 13 belong to the same subfamily of Rab-GTPases. Here we show that insulin promotes GTP loading of Rab13 and Rab8A but not Rab10 in rat L6 muscle cells, Rab8A activation preceding that of Rab13. siRNA-mediated Rab13 knockdown blocked the insulin-induced increase of GLUT4 at the muscle cell surface that was rescued by a Rab13 ortholog but not by Rab8A. Constitutively active AS160 lowered basal and insulin-stimulated levels of surface GLUT4, effects that were reversed by overexpressing Rab8A or Rab13, suggesting that both Rabs are targets of AS160-GAP activity in the context of GLUT4 traffic. Rab13 had a broader intracellular distribution compared with the perinuclear restriction of Rab8A, and insulin promoted Rab13 colocalization with GLUT4 at the cell periphery. We conclude that Rab13 and Rab8A are Rab-GTPases activated by insulin, and that downstream of AS160 they regulate traffic of GLUT4 vesicles, possibly acting at distinct steps and sites. These findings close in on the series of events regulating muscle GLUT4 traffic in response to insulin, crucial for whole-body glucose homeostasis.     

From the Cover: A Rab GAP cascade defines the boundary between two Rab GTPases on the secretory pathway

Membrane traffic along the endocytic and exocytic pathways relies on the appropriate localization and activation of a series of different Rab GTPases. Rabs are activated by specific guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). GEF cascades, in which one Rab in its GTP-bound form recruits the GEF that activates the next Rab along the pathway, can account for the sequential activation of a series of Rabs, but it does not explain how the first Rab is inactivated after the next Rab has been activated. We present evidence for a counter-current GAP cascade that serves to restrict the spatial and temporal overlap of 2 Rabs, Ypt1p and Ypt32p, on the exocytic pathway in Saccharomyces cerevisiae. We show that Gyp1p, a GAP for Ypt1p, specifically interacts with Ypt32p, and that this interaction is important for the localization and stability of Gyp1p. Moreover, we demonstrate that, in WT cells, Ypt1p compartments are converted over time into Ypt32p compartments, whereas in gyp1Δ cells there is a significant increase in compartments containing both proteins that reflects a slower transition from Ypt1p to Ypt32p. GEF cascades working in concert with counter-current GAP cascades could generate a programmed series of Rab conversions responsible for regulating the choreography of membrane traffic.     

Identification of a nucleotide exchange-promoting activity for p21ras.

The biological activity of proteins encoded by the ras family of oncogenes is dependent on whether they are bound to GTP or GDP: the type of nucleotide bound is dependent on the rate of GTP hydrolysis (promoted by the GTPase-activating protein, GAP) and the rate of nucleotide exchange with cytosolic pools. A protein that stimulates the rate of exchange of guanine nucleotide on p21ras has been identified and characterized in cytoplasmic extracts of human placenta. The exchange-promoting protein runs on a gel filtration column with an apparent relative molecular weight of about 60,000. It is sensitive to heat and to trypsin. The exchange-promoting protein acts reversibly and does not cause degradation of p21ras. It is inactive towards the alpha subunit of a heterotrimeric GTP-binding protein (Go alpha) but acts on a large number of different mutant ras proteins, including transforming and effector mutants that are insensitive to the action of GAP. This protein, which we have termed REP (ras exchange-promoting), has the characteristics expected of a physiological activator of p21ras in cellular growth-signal-transduction pathways.     

Role of rab GDP dissociation inhibitor alpha in regulating plasticity of hippocampal neurotransmission

Rab GDP dissociation inhibitor alpha (Rab GDIalpha) is a regulator of the Rab small G proteins implicated in neurotransmission, and mutations of Rab GDIalpha cause human X-linked mental retardation associated with epileptic seizures. In Rab GDIalpha-deficient mice, synaptic potentials in the CA1 region of the hippocampus displayed larger enhancement during repetitive stimulation, which was apparently opposite to the phenotype of Rab3A-deficient mice. Furthermore, the Rab GDIalpha-deficient mice showed hypersensitivity to bicuculline, an inducer of epileptic seizures. These results suggest that Rab GDIalpha plays a specialized role in Rab3A recycling to suppress hyperexcitability via modulation of presynaptic forms of plasticity.     

Peptide Chain Termination: Effect of Protein S on Ribosomal Binding of Release Factors

The protein factor S, previously shown to stimulate polypeptide chain termination in bacterial extracts, has two effects upon the complex formed between ribosomes, release factor, and terminator (trinucleotide) codon: (1) in the absence of GTP or GDP, S stimulates formation of an [R·UAA·ribosome] intermediate, and (2) in the presence of GTP or GDP, S participates in dissociation of this intermediate. Factor S can stimulate fMet release from [fMet-tRNA^f·AUG·ribosome] intermediates in either the presence or absence of GTP or GDP. A model is proposed which relates the in vitro effects of S ± GTP (or GDP) on fMet release to the effects of S ± GTP (or GDP) on the binding and dissociation of R factor from ribosomes.