The amphioxus rab GDP-dissociation inhibitor (GDI) gene is neural-specific: implications for the evolution of chordate rab GDI genes.

The rab GDP-dissociation inhibitor (rab GDI) proteins are involved in the regulation of vesicle-mediated cellular transport. We isolated the amphioxus rab GDI gene, analyzed its expression during amphioxus development, and performed a phylogenetic analysis of the rab GDI family. In contrast to the two major rab GDI forms in mammals, the alpha and beta forms, there is only one rab GDI isoform in amphioxus. Our analysis indicates that the occurrence of the alpha and beta forms of rab GDI preceded the divergence of lineages leading to birds and mammals, and that the amphioxus rab GDI may have evolved directly from the common ancestor of both forms. While the mammalian rab GDI beta-genes are ubiquitously expressed, the rab GDI alpha genes are predominantly expressed in neural tissues. The expression analysis of the amphioxus rab GDI gene shows predominantly neural expression similar to that of the mammalian rab GDI alpha form, suggesting that the ancestral expression pattern of chordate rab GDI was neural. In addition, the chicken rab GDI beta-like gene also shows neural-specific expression, which indicates that the neural expression was retained in both early postduplication alpha and beta isoforms and that a novel function associated with ubiquitous expression may have evolved uniquely in mammals. These results reveal a likely scenario of functional divergence of the rab GDI genes after duplication of the ancestral gene. A similar pattern of evolution, in which one of the duplicated genes retained a role similar to that of the ancestral one while other genes were recruited into novel roles, was also observed in the analysis of chordate Otx and hedgehog genes. In the rab GDI, hedgehog, and Otx gene families, the gene retaining the ancestral role shows a lower rate of sequence evolution than its counterpart, which was recruited for a novel function.     

A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons.

One of the major activities of developing neurons is the transport of new membrane to the growing axon. Candidates for playing a key role in the regulation of this intense traffic are the small GTP-binding proteins of the rab family. We have used hippocampal neurons in culture and analyzed membrane traffic activity after suppressing the expression of the small GTP-binding protein rab8. Inhibition of protein expression was accomplished by using sequence-specific antisense oligonucleotides. While rab8 depletion resulted in the blockage of morphological maturation in 95% of the neurons, suppression of expression of another rab protein, rab3a, had no effect, and all neurons developed normal axons and dendrites. The impairment of neuronal maturation by rab8 antisense treatment was due to inhibition of membrane traffic. Thus, by using video-enhanced differential interference contrast microscopy, we observed in the rab8-depleted cells a dramatic reduction in the number of vesicles undergoing anterograde transport. Moreover, by incubating antisense-treated neurons with Bodipy-labeled ceramide, a fluorescent marker for newly formed exocytic vesicles, we observed fluorescence labeling restricted to the Golgi apparatus, whereas in control cells labeling was found also in the neurites. These results show the role of the small GTPase rab8 in membrane traffic during neuronal process outgrowth.     

Manipulation of rab GTPase function by intracellular bacterial pathogens.

Intracellular bacterial pathogens have evolved highly specialized mechanisms to enter and survive within their eukaryotic hosts. In order to do this, bacterial pathogens need to avoid host cell degradation and obtain nutrients and biosynthetic precursors, as well as evade detection by the host immune system. To create an intracellular niche that is favorable for replication, some intracellular pathogens inhibit the maturation of the phagosome or exit the endocytic pathway by modifying the identity of their phagosome through the exploitation of host cell trafficking pathways. In eukaryotic cells, organelle identity is determined, in part, by the composition of active Rab GTPases on the membranes of each organelle. This review describes our current understanding of how selected bacterial pathogens regulate host trafficking pathways by the selective inclusion or retention of Rab GTPases on membranes of the vacuoles that they occupy in host cells during infection.     

Rap1 GTPase inhibits leukocyte transmigration by promoting endothelial barrier function

The passage of leukocytes out of the blood circulation and into tissues is necessary for the normal inflammatory response, but it also occurs inappropriately in many pathological situations. This process is limited by the barrier presented by the junctions between adjacent endothelial cells that line blood vessels. Here we show that activation of the Rap1 GTPase in endothelial cells accelerated de novo assembly of endothelial cell-cell junctions and increased the barrier function of endothelial monolayers. In contrast, depressing Rap1 activity by expressing Rap1GAP led to disassembly of these junctions and increased their permeability. We also demonstrate that endogenous Rap1 was rapidly activated at early stages of junctional assembly, confirming the involvement of Rap1 during junctional assembly. Intriguingly, elevating Rap1 activity selectively within endothelial cells decreased leukocyte transendothelial migration, whereas inhibiting Rap1 activity by expression of Rap1GAP increased leukocyte transendothelial migration, providing physiological relevance to our hypothesis that Rap1 augments barrier function of inter-endothelial cell junctions. Furthermore, these results suggest that Rap1 may be a novel therapeutic target for clinical conditions in which an inappropriate inflammatory response leads to disease.     

The membrane-bound GTPase Guf1 promotes mitochondrial protein synthesis under suboptimal conditions

Recently, the bacterial elongation factor LepA was identified as critical for the accuracy of in vitro translation reactions. Extremely well conserved homologues of LepA are present throughout bacteria and eukaryotes, but the physiological relevance of these proteins is unclear. Here we show that the yeast counterpart of LepA, Guf1, is located in the mitochondrial matrix and tightly associated with the inner membrane. It binds to mitochondrial ribosomes in a GTP-dependent manner. Mutants lacking Guf1 show cold- and heat-sensitive growth defects on non-fermentable carbon sources that are especially pronounced under nutrient-limiting conditions. The cold sensitivity is explained by diminished rates of protein synthesis at low temperatures. At elevated temperatures, Guf1-deficient mutants exhibit defects in the assembly of cytochrome oxidase, suggesting that the polypeptides produced are not functional. Moreover, Guf1 mutants exhibit synthetic growth defects with mutations of the protein insertase Oxa1. These observations show a critical role for Guf1 in vivo. The observed defects in Guf1-deficient mitochondria are consistent with a function of Guf1 as a fidelity factor of mitochondrial protein synthesis.     

The Golgi matrix protein GM130: a specific interacting partner of the small GTPase rab1b

To detect specific partners of the small Golgi-localized GTPase rab1b we generated rab1b mutants and used them as bait proteins in yeast two-hybrid screens. We isolated several specifically interacting clones. Two of them encode large protein fragments highly homologous to rat GM130 and to human Golgin95. The full-length human GM130 cDNA was cloned and its interaction with rab1b was characterized in detail by yeast two-hybrid and in vitro binding assays. Here we report for the first time that the rab1b protein interacts specificially with GM130 in a GTP-dependent manner and therefore needs the hypervariable regions of the N- and C-termini. We mapped the rab1b binding site of GM130 and provide evidence that it is different to the previously described p115 and Grasp65 binding sites of the GM130 protein.     

Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.

Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.     

Regulation of the GTPase activity of the ras-like protein p25rab3A. Evidence for a rab3A-specific GAP.

The rab3A gene product is a 25-kilodalton guanine nucleotide-binding protein, expressed at high levels in neural tissue, which has about 30% homology to ras. Recombinant rab3A protein and p25rab3A purified from bovine brain membranes have been used as substrates to look for factors that regulate its biochemical activity. A detergent-soluble factor associated with rat brain membranes exists that accelerates the GTPase activity of both mammalian and recombinant p25rab3A. The activity was thermolabile, sensitive to trypsin, and behaved like an integral membrane protein. GTPase-activating protein (GAP) activity toward p25rab3A was also detected in the cytosolic fraction. This activity was observed in all other tissues examined, in addition to brain. Based upon dose-response data, the rab3A-GAP activity from rat brain was approximately equally distributed between cytosolic and membrane fractions; no activity was found in the nuclear fraction. Recombinant ras-specific GAP had no effect upon the GTPase activity of p25rab3A. By gel filtration chromatography, the factor in rat brain cytosol has a molecular size of 400,000 daltons.     

Impaired recruitment of the small GTPase rab7 correlates with the inhibition of phagosome maturation by Leishmania donovani promastigotes

We have shown recently that one of the survival strategies used by Leishmania donovani promastigotes during the establishment of infection in macrophages consists in inhibiting phagosome–endosome fusion. This inhibition requires the expression of lipophosphoglycan (LPG), the predominant surface glycoconjugate of promastigotes, as parasites expressing truncated forms of LPG reside in phagosomes that fuse extensively with endocytic organelles. In the present study, we developed a single-organelle fluorescence analysis approach to study and analyse the intracellular trafficking of 'fusogenic' and 'low-fusogenic' phagosomes induced by an LPG repeating unit-defective mutant ( lpg2 KO) or by wild-type L. donovani promastigotes respectively. The results obtained indicate that phagosomes containing mutant parasites fuse extensively with endocytic organelles and transform into phagolysosomes by losing the early endosome markers EEA1 and transferrin receptor, and acquiring the late endocytic and lysosomal markers rab7 and LAMP1. In contrast, a majority of 'low-fusogenic' phagosomes containing wild-type L. donovani promastigotes do not acquire rab7, wheres they acquire LAMP1 with slower kinetics. These results suggest that L. donovani parasites use LPG to restrict phagosome–endosome fusion at the onset of infection in order to prevent phagosome maturation. This is likely to permit the transformation of hydrolase-sensitive promastigotes into hydrolase-resistant amastigotes within a hospitable vacuole not displaying the harsh environment of phagolysosomes.     

Biochemical properties of the YPT-related rab1B protein. Comparison with rab1A

We recently identified a novel rat cDNA: rab1B, closely related to the rab1A cDNA and to the yeast YPT1 gene. The rab1B cDNA encodes a 202 amino acid protein (22.1 kDa) that was produced in Escherichia coli under the control of the phi 10 promoter for the T7 RNA polymerase. The rab1B protein was purified in large amounts to near homogeneity in a simplified procedure. We studied the biochemical properties of rab1B and rab1A proteins. They both bind specifically GTP and GDP and possess intrinsic GTPase activities. The rab1B Lys21----Met mutant protein does not bind GTP, whereas the Ala65----Thr mutant has a reduced GTPase activity and is competent for autophosphorylation in the presence of GTP.     

The rab7 GTPase controls the maturation of Salmonella typhimurium-containing vacuoles in HeLa cells.

Following entry into non-phagocytic HeLa cells, the facultative pathogen Salmonella typhimurium survives and replicates within a membrane-bound vacuole. Preceding the initiation of intracellular replication there is a lag phase, during which the bacteria modulate their environment. This phase is characterized by the rapid recycling of early endosomal proteins present on the nascent vacuole followed by the acquisition of a subset of lysosomal proteins. To gain a better understanding of the mechanism of intracellular survival, we have followed the biogenesis of the S. typhimurium-containing vacuole (SCV) in HeLa cells expressing different mutant forms of the small GTPase rab7. We demonstrate that the SCV recruits pre-existing lysosomal glycoproteins (Lgps) in a rab7-dependent manner, without directly interacting with lysosomes. We also show the transient accumulation, in the vicinity of the SCV, of novel rab7- and Lgp-containing vesicles containing very low amounts of cathepsin D. The size of these vesicles is dependent on rab7 activity, suggesting a role for rab7 in their homotypic fusion. Taken together, these results indicate that rab7 regulates SCV biogenesis during the phase characterized by the rapid acquisition of lysosomal proteins. We propose that SCV maturation involves its interaction with rab7/Lgp-containing vesicles which are possible intermediate cargo components of the late endocytic pathway.     

A structural model of the GDP dissociation inhibitor rab membrane extraction mechanism

Rab GDP dissociation inhibitors (GDI)-facilitated extraction of prenylated Rab proteins from membranes plays an important role in vesicular membrane trafficking. The investigated thermodynamic properties of yeast Rab.GDI and Rab.MRS6 complexes demonstrated differences in the Rab binding properties of the closely related Rab GDI and MRS6 proteins, consistent with their functional diversity. The importance of the Rab C terminus and its prenylation for GDI/MRS6 binding was demonstrated using both biochemical and structural data. The presented structures of the apo-form yeast Rab GDI and its two complexes with unprenylated Rab proteins, together with the earlier published structures of the prenylated Ypt1.GDI, provide evidence of allosteric regulation of the GDI lipid binding site opening, which plays a key role in the proposed mechanism of GDI-mediated Rab extraction. We suggest a model for the interaction of GDI with prenylated Rab proteins that incorporates a stepwise increase in affinity as the three different partial interactions are successively formed.     

The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.

We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5-ile133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.     

A rab1 GTPase is required for transport between the endoplasmic reticulum and golgi apparatus and for normal golgi movement in plants

We describe a green fluorescent protein (GFP)-based assay for investigating membrane traffic on the secretory pathway in plants. Expression of AtRab1b(N121I), predicted to be a dominant inhibitory mutant of the Arabidopsis Rab GTPase AtRab1b, resulted in accumulation of a secreted GFP marker in an intracellular reticulate compartment reminiscent of the endoplasmic reticulum. This accumulation was alleviated by coexpressing wild-type AtRab1b but not AtRab8c. When a Golgi-targeted and N-glycosylated variant of GFP was coexpressed with AtRab1b(N121I), the variant also accumulated in a reticulate network and an endoglycosidase H-sensitive population appeared. Unexpectedly, expression of AtRab1b(N121I), but not of the wild-type AtRab1b, resulted in a reduction or cessation of vectorial Golgi movement, an effect that was reversed by coexpression of the wild type. We conclude that AtRab1b function is required for transport from the endoplasmic reticulum to the Golgi apparatus and suggest that this process may be coupled to the control of Golgi movement.     

Extracellular Superoxide Dismutase Regulates the Expression of Small GTPase Regulatory Proteins GEFs,GAPs, and GDI

Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3–induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3–driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme.     

Structure of the GTPase and GDI domains of FeoB, the ferrous iron transporter of Legionella pneumophila

Prokaryotic pathogens have developed specialized mechanisms for efficient uptake of ferrous iron (Fe²⁺) from the host. In Legionella pneumophila, the causative agent of Legionnaires’ disease, the transmembrane GTPase FeoB plays a key role in Fe²⁺ acquisition and virulence. FeoB consists of a membrane-embedded core and an N-terminal, cytosolic region (NFeoB). Here, we report the crystal structure of NFeoB from L. pneumophila, revealing a monomeric protein comprising two separate domains with GTPase and guanine-nucleotide dissociation inhibitor (GDI) functions. The GDI domain displays a novel fold, whereas the overall structure of the GTPase domain resembles that of known G domains but is in the rarely observed nucleotide-free state.     

Mislocalization or reduced expression of Arf GTPase-activating protein ASAP1 inhibits cell spreading and migration by influencing Arf1 GTPase cycling

ADP-ribosylation factor (Arf) family of small GTP-binding proteins plays a central role in membrane trafficking and cytoskeletal remodeling. ASAP1 (Arf-GAP containing SH3, ankyrin repeats, and PH domain) is a phospholipid-dependent Arf GTPase-activating protein (Arf-GAP) that binds to protein-tyrosine kinases Src and focal adhesion kinase. Using affinity chromatography and mass spectrometry (MS), we identified the adaptor protein CD2-associated protein (CD2AP) as a candidate binding partner of ASAP1. Both co-immunoprecipitation and GST pull-down experiments confirmed that CD2AP stably interacts with ASAP1 through its N-terminal SH3 domains. Using a mislocalization strategy, we show that sequestration of endogenous ASAP1 to mitochondria with a CD2AP SH3-mito fusion protein (the three N-terminal SH3 domains of CD2AP fused to Listeria monocytogenes ActA mitochondria-targeting sequence) inhibited REF52 cell spreading and migration in response to fibronectin stimulation. Using an alternative strategy we show that suppressing ASAP1 expression with small interfering RNA duplexes also significantly retarded cell spreading and inhibited cell migration. Furthermore, abrogation of ASAP1 function using either small interfering RNAs or mislocalization approaches caused an increase of GTP loading on Arf1 and loss of paxillin from adhesions. These results taken together with our previous observations that overexpression of ASAP1 inhibits cell spreading and alters paxillin localization to adhesions (Liu, Y., Loijens, J. C., Martin, K. H., Karginov, A. V., and Parsons, J. T. (2002) Mol. Biol. Cell. 13, 2147-2156) suggest that the recruitment of certain adhesion components such as paxillin requires dynamic GTP/GDP turnover of Arf1 GTPase.     

Golgi recruitment of GRIP domain proteins by Arf-like GTPase 1 is regulated by Arf-like GTPase 3

Golgins are Golgi-localized proteins present in all molecularly characterized eukaryotes that function in Golgi transport and maintenance of Golgi structure. Some peripheral membrane Golgins, including the yeast Imh1 protein, contain the recently described GRIP domain that can independently mediate Golgi localization by an unknown mechanism. To identify candidate Golgi receptors for GRIP domain proteins, a collection of Saccharomyces cerevisiae deletion mutants was visually screened by using yeast, mouse, and human GFP-GRIP domain fusion proteins for defects in Golgi localization. GFP-GRIP reporters were localized to the cytosol in cells lacking either of two ARF-like (ARL) GTPases, Arl1p and Arl3p. In vitro binding experiments demonstrated that activated Arl1p-GTP binds specifically and directly to the Imh1p GRIP domain. Arl1p colocalized with Imh1p-GRIP at the Golgi, and Golgi localization of Arl1p was regulated by the GTPase cycle of Arl3p. These results suggest a cascade in which the GTPase cycle of Arl3p regulates Golgi localization of Arl1p, which in turn binds to the GRIP domain of Imh1p and recruits it to the Golgi. The similar requirements for localization of GRIP domains from yeast, mouse, and human when expressed in yeast, and the presence of Arl1p and Arl3p homologs in these species, suggest that this is an evolutionarily conserved mechanism.     

Spatial regulation of the exocyst complex by Rho1 GTPase

Spatial regulation of membrane traffic is fundamental to many biological processes, including epithelial cell polarization and neuronal synaptogenesis. The multiprotein exocyst complex is localized to sites of polarized exocytosis, and is required for vesicle targeting and docking at specific domains of the plasma membrane. One component of the complex, Sec3, is thought to be a spatial landmark for polarized exocytosis. We have searched for proteins that regulate the polarized localization of the exocyst in the budding yeast Saccharomyces cerevisiae. Here we report that certain rho1 mutant alleles specifically affect the localization of the exocyst proteins. Sec3 interacts directly with Rho1 in its GTP-bound form, and functional Rho1 is needed both to establish and to maintain the polarized localization of Sec3. Sec3 is not the only mediator of the effect of Rho1 on the exocyst, because some members of the complex are correctly targeted independently of the interaction between Rho1 and Sec3. These results reveal the action of parallel pathways for the polarized localization of the exocytic machinery, both of which are under the control of Rho1, a master regulator of cell polarity.