Rab GTPases: specifying and deciphering organelle identity and function

Ten years ago, 20 Rab proteins had been identified as organelle-specific GTPases, and two were known to be essential for vesicle targeting in yeast. Today, more than 60 mammalian Rab proteins have been identified. While Rabs were always viewed as key regulatory factors, no one could have anticipated their diversity of functions and multitude of effectors. Rabs organize distinct protein scaffolds within a single organelle and act in a combinatorial manner with their effectors to regulate all stages of membrane traffic.     

An Overexpression Screen of Toxoplasma gondii Rab-GTPases Reveals Distinct Transport Routes to the Micronemes

The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.     

The Rab GTPase family

The Rab family is part of the Ras superfamily of small GTPases. There are at least 60 Rab genes in the human genome, and a number of Rab GTPases are conserved from yeast to humans. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane traffic pathways. In the GTP-bound form, the Rab GTPases recruit specific sets of effector proteins onto membranes. Through their effectors, Rab GTPases regulate vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion.     

Rab 蛋白调控胞内囊泡运输

细胞内各个细胞器之间通过囊泡的膜转运是真核细胞存在的基本。Rab蛋白确保了转运蛋白被运输至正确的目的地。Rab蛋白是小GTP酶中的一大家族,它通过募集其效应物蛋白,其中包括接头蛋白,栓系因子,激酶,磷酸酶以及动力蛋白等,调控了细胞膜的选取,囊泡出芽,去包被,转运以及膜融合等过程。本文主要从Rab蛋白循环着手,依次论述了Rab蛋白在囊泡出芽,去包被,转运和膜融合等过程中起到的作用,从而使读者对Rab蛋白能有一个更加系统的了解。     

Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.     

Protein-targeting determinants in the secretory pathway of apicomplexan parasites

Apicomplexan parasites possess a highly specialized secretory apparatus. The timed secretion of proteins from three different organelles--micronemes, rhoptries and dense granules--serves to establish and maintain a parasitophorous vacuole inside the host cell in which the parasites can divide. Recent efforts have identified components that sort apicomplexan proteins to these unusual secretory organelles and have shown that this machinery is evolutionarily conserved across species. Concise amino acid sequences (e.g. tyrosine-based motifs) within the targeted protein determine their destination in Apicomplexa in a way similar to mammalian cells. Additionally, the parasite exploits new or unusual mechanisms of protein targeting (e.g. post-secretory membrane insertion).     

The functions of Rab GTPases in plant membrane traffic

Rab GTPases are important determinants of membrane identity and membrane targeting. Higher plants have evolved a unique set of Rab GTPases that presumably reflects the specific demands of plant cell trafficking. In recent years, significant progress has been made in identifying Rab GTPases involved in endosome organisation, cytokinesis and in post-Golgi traffic to the plasma membrane and vacuoles. These include members of the Rab-F1, Rab-F2, Rab-A1, Rab-A2 and Rab-A4 subclasses. Some important regulators or effectors have also been identified for Rab-F, Rab-A1 and Rab-A4 proteins. However, uncertainties remain about the trafficking pathways that connect the compartments in the trans-Golgi/prevacuolar/endosomal system and there is still little or no insight into the functions of several major subclasses within the Rab GTPase family.     

A novel statin-mediated “prenylation block-and-release” assay provides insight into the membrane targeting mechanisms of small GTPases

Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a ‘prenylation block-and-release’ assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.     

Small GTPase Rab21 regulates cell adhesion and controls endosomal traffic of beta1-integrins

Dynamic turnover of integrin cell adhesion molecules to and from the cell surface is central to cell migration. We report for the first time an association between integrins and Rab proteins, which are small GTPases involved in the traffic of endocytotic vesicles. Rab21 (and Rab5) associate with the cytoplasmic domains of alpha-integrin chains, and their expression influences the endo/exocytic traffic of integrins. This function of Rab21 is dependent on its GTP/GDP cycle and proper membrane targeting. Knock down of Rab21 impairs integrin-mediated cell adhesion and motility, whereas its overexpression stimulates cell migration and cancer cell adhesion to collagen and human bone. Finally, overexpression of Rab21 fails to induce cell adhesion via an integrin point mutant deficient in Rab21 association. These data provide mechanistic insight into how integrins are targeted to intracellular compartments and how their traffic regulates cell adhesion.     

Signal transduction gRABs attention

Rab proteins are small GTPases involved in the regulation of vesicular membrane traffic. Research done in the past years has demonstrated that some of these proteins are under the control of signal transduction pathways. Still, several recent papers point out to a new unexpected role for this family of Ras-related proteins, as potential regulators of intracellular signaling pathways. In particular, several evidence indicate that members of the Rab family of small GTPases, through their effectors, are key molecules participating to the regulation of numerous signal transduction pathways profoundly influencing cell proliferation, cell nutrition, innate immune response, fragmentation of compartments during mitosis and apoptosis. Even more surprisingly, direct involvement of Rab proteins in signaling to the nucleus has been demonstrated. This review will focus on aspects of Rab proteins function connected to signal transduction and, in particular, connections between membrane traffic and other cell pathways will be examined.     

Dissection of minimal sequence requirements for rhoptry membrane targeting in the malaria parasite

Rhoptries are specialized secretory organelles characteristic of single cell organisms belonging to the clade Apicomplexa. These organelles play a key role in the invasion process of host cells by accumulating and subsequently secreting an unknown number of proteins mediating host cell entry. Despite their essential role, little is known about their biogenesis, components and targeting determinants. Here, we report on a conserved apicomplexan protein termed Armadillo Repeats-Only (ARO) protein that we localized to the cytosolic face of Plasmodium falciparum and Toxoplasma gondii rhoptries. We show that the first 20 N-terminal amino acids are sufficient for rhoptry membrane targeting. This protein relies on both - myristoylation and palmitoylation motifs - for membrane attachment. Although these lipid modifications are essential, they are not sufficient to direct ARO to the rhoptry membranes. Mutational analysis revealed additional residues within the first 20 amino acids of ARO that play an important role for rhoptry membrane attachment: the positively charged residues R9 and K14. Interestingly, the exchange of R9 with a negative charge entirely abolishes membrane attachment, whereas the exchange of K14 (and to a lesser extent K16) alters only its membrane specificity. Additionally, 17 proteins predicted to be myristoylated and palmitoylated in the first 20 N-terminal amino acids were identified in the genome of the malaria parasite. While most of the corresponding GFP fusion proteins were trafficked to the parasite plasma membrane, two were sorted to the apical organelles. Interestingly, these proteins have a similar motif identified for ARO.     

Multiple aspects of Rab protein action in the secretory pathway: focus on Rab3 and Rab6

Rab proteins form the largest branch of the Ras superfamily of GTPases. They are localized to the cytoplasmic face of organelles and vesicles involved in the biosynthetic/secretory and endocytic pathways in eukaryotic cells. It is now well established that Rab proteins play an essential role in the processes that underlie the targeting and fusion of transport vesicles with their appropriate acceptor membranes. They perform this task through interactions with a wide variety of effector molecules. In this review, we illustrate recent advances in the field of Rab GTPases, taking as examples two proteins involved in the biosynthetic pathway, Rab3 and Rab6.     

Sitting in the driver's seat: Manipulation of mammalian cell Rab GTPase functions by apicomplexan parasites

Many intracellular microbial pathogens subvert, disrupt or otherwise modulate host membrane trafficking pathways to establish a successful infection. Among them, bacteria that are trapped in a phagosome during mammalian cell invasion, disengage the programmed degradation process by altering the identity of their replicative niche through the exclusion or recruitment of specific Rab GTPases to their vacuole. Many viruses co-opt essential cellular trafficking pathways to perform key steps in their lifecycles. Among protozoan parasites, Apicomplexa are obligate intracellular microbes that invade mammalian cells by creating a unique, nonfusogenic membrane-bound compartment that protects the parasites straightaway from lysosomal degradation. Recent compelling evidence demonstrates that apicomplexan parasites are master manipulators of mammalian Rab GTPase proteins, and benefit or antagonise Rab functions for development within host cells. This review covers the exploitation of mammalian Rab proteins and vesicles by Apicomplexa, focusing on Toxoplasma, Neospora, Plasmodium and Theileria parasites.     

Rab3A and Rab3D control the total granule number and the fraction of granules docked at the plasma membrane in PC12 cells

Rab proteins are Ras-like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immuno-electron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild-type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.     

Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells

Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells. Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.     

Functionality and specific membrane localization of transport GTPases carrying C-terminal membrane anchors of synaptobrevin-like proteins.

Ras-related guanine nucleotide-binding proteins of the Ypt/Rab family fulfill a pivotal role in vesicular protein transport both in yeast and in mammalian cells. Proper functioning of these proteins involves their cycling between a GTP- and a GDP-bound state as well as their reversible association with specific membranes. Here we show that the yeast Ypt1 and Sec4 proteins, essential components of the vesicular transport machinery, allow unimpaired vesicular transport when permanently fixed to membranes by membrane-spanning domains replacing their two C-terminal cysteine residues. Membrane detachment of the GTPases therefore is not obligatory for transport vesicle docking to or fusion with an acceptor membrane. It was also found that the membrane anchors derived from different synaptobrevin-related proteins have targeting information and direct the chimeric GTPases to different cellular compartments, presumably from the endoplasmic reticulum via the secretory pathway.     

The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells.

There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic along biosynthetic and endocytotic pathways in eukaryotic cells. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. We report here that overexpression in PC12 cells of Rab3a mutant proteins defective in either GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. Finally, the electrophysiological approach showed that bovine chromaffin cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. In contrast, control cells showed a phenomenon of desensitization. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis from occurring unless secretion is triggered. Furthermore, it is proposed that Rab3a is involved in adaptive processes such as response habituation.     

Location, location, location: trafficking and function of secreted proteases of Toxoplasma and Plasmodium

The Apicomplexan parasites Toxoplasma gondii and Plasmodium species are obligate intracellular parasites that rely upon unique secretory organelles for invasion and other specialized functions. Data is emerging that proteases are critical for the biogenesis of micronemes and rhoptries, regulated secretory organelles reminiscent of dense core granules and secretory lysosomes of higher eukaryotes. Proteases targeted to the Plasmodium food vacuole, a unique organelle dedicated to hemoglobin degradation, are also critical to parasite survival. Thus study of the targeting and function of the proteases of the Apicomplexa provides a fascinating model system to understand regulated secretion and secretory organelle biogenesis.     

Rac1‐Rab11‐FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T‐cell activation

The immunological synapse generation and function is the result of a T‐cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11‐positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1‐dependent manner, key morphological events, like T‐cell spreading and synapse symmetry. Finally, Rab11‐/FIP3‐mediated regulation is necessary for T‐cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T‐cell activation.