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.     

Rab6 and the secretory pathway affect oocyte polarity in Drosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFalpha homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.     

Rab GTPases and microtubule motors

Rab proteins are a family of small GTPases which, since their initial identification in the late 1980s, have emerged as master regulators of all stages of intracellular trafficking processes in eukaryotic cells. Rabs cycle between distinct conformations that are dependent on their guanine-nucleotide-bound status. When active (GTP-bound), Rabs are distributed to the cytosolic face of specific membranous compartments where they recruit downstream effector proteins. Rab-effector complexes then execute precise intracellular trafficking steps, which, in many cases, include vesicle motility. Microtubule-based kinesin and cytoplasmic dynein motor complexes are prominent among the classes of known Rab effector proteins. Additionally, many Rabs associate with microtubule-based motors via effectors that act as adaptor molecules that can simultaneously associate with the GTP-bound Rab and specific motor complexes. Thus, through association with motor complexes, Rab proteins can allow for membrane association and directional movement of various vesicular cargos along the microtubule cytoskeleton. In this mini-review, we highlight the expanding repertoire of Rab/microtubule motor protein interactions, and, in doing so, present an outline of the multiplicity of transport processes which result from such interactions.     

Golgi-bound Rab34 is a novel member of the secretory pathway

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.     

Controlling the location and activation of Rab GTPases

The remarkable degree of specificity with which Rab GTPases recognise distinct subsets of intracellular membranes forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the right membrane at the right time. The molecular mechanisms controlling Rab localisation, however, have proved tricky issues to address. It is becoming increasingly apparent that multiple factors contribute to the specificity of Rab localisation and the close coordination of membrane targeting with Rab activation. With important new insights into the mode of action of the general Rab regulators REP and RabGDI, as well as the demonstration that novel factors such as Yip3/Pra1 act as GDI displacement factors and that signals within Rab proteins contribute to targeting specificity, a better understanding of the concepts governing Rab recruitment and function is now beginning to emerge. The diversity of cellular processes regulated by Rab family members is made possible, not only by the wide range of effectors they recruit, but also by the different mechanisms regulating their own targeting and activation.     

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。     

植物Rab蛋白的分类与功能

Rab蛋白构成小G蛋白超家族中最大的1个家族,广泛存在于动物、植物和微生物中.Rab调控细胞内的囊泡形成、转运、锚定及囊泡与质膜的融合等过程,在细胞内吞和分泌途径中发挥分子开关的作用.不同生物中Rab的结构和作用机制非常保守,但Rab的分类和生理学功能存在差异.植物Rab不仅行使类似于动物或微生物同源Rab的细胞学功能,而且在植物生长发育、激素信号调节、生物或非生物胁迫应答等方面表现出功能特异性.本文结合近年的研究进展,对植物Rab的分类、结构、调节机制和功能进行了综述,并对当前植物Rab功能研究的难点和方向进行了
讨论.     

Role of endosomal Rab GTPases in cytokinesis

Completion of cytokinesis requires Rab 11-dependent membrane trafficking events to deliver new membrane to the furrow and for abscission. Many Rabs have overlapping endosomal distributions, hence, we examined whether these Rabs also function in cytokinesis. Analysis of the distribution of Rabs 4, 5, 7, 8, 9, 11, 21, and 22 revealed that only Rab 11 was enriched within the furrow of cells in telophase or present within the midbody. By contrast, Rabs 4, 5, 7, 8, and 9 were mainly localised within a peri-nuclear compartment facing away from the furrow. Using RNA interference and dominant negative Rab mutants, we evaluated the role of these Rabs in furrowing and abscission. Consistent with previous work, we find that Rab 11 is intimately involved in abscission. However, we further found that depletion of Rab 4 slowed but did not prevent abscission. Depletion of any other Rab species had little effect on furrowing or abscission. These data suggest that the membrane trafficking events required for completion of cytokinesis are largely controlled by Rab 11 and not other endosomal Rab proteins, and further suggest that the relocation of Rab 11-specific cargo is an integral facet of abscission. Arf6 knockdown was without effect on cytokinesis, but when both Rab 11 and Arf6 were knocked-down, we found the furrow rapidly regressed and the cells were unable to form a stable midbody. We suggest that Rab 11 and Arf6 function synergistically in the switch from furrowing to abscission, as well as in the terminal stage of abscission.     

Regulation of mTORC1 by the Rab and Arf GTPases

The mammalian target of rapamycin (mTOR) is a key cell growth regulator, which forms two distinct functional complexes (mTORC1 and mTORC2). mTORC1, which is directly inhibited by rapamycin, promotes cell growth by stimulating protein synthesis and inhibiting autophagy. mTORC1 is regulated by a wide range of extra- and intracellular signals, including growth factors, nutrients, and energy levels. Precise regulation of mTORC1 is important for normal cellular physiology and development, and dysregulation of mTORC1 contributes to hypertrophy and tumorigenesis. In this study, we screened Drosophila small GTPases for their function in TORC1 regulation and found that TORC1 activity is regulated by members of the Rab and Arf family GTPases, which are key regulators of intracellular vesicle trafficking. In mammalian cells, uncontrolled activation of Rab5 and Arf1 strongly inhibit mTORC1 activity. Interestingly, the effect of Rab5 and Arf1 on mTORC1 is specific to amino acid stimulation, whereas glucose-induced mTORC1 activation is not blocked by Rab5 or Arf1. Similarly, active Rab5 selectively inhibits mTORC1 activation by Rag GTPases, which are involved in amino acid signaling, but does not inhibit the effect of Rheb, which directly binds and activates mTORC1. Our data demonstrate a key role of Rab and Arf family small GTPases and intracellular trafficking in mTORC1 activation, particularly in response to amino acids.     

Rab GTPases implicated in inherited and acquired disorders

The endocytotic machinery imports, transports and exports receptors and associated molecules between the plasma membrane and various cytoplasmic chambers resulting in selective recycling, degradation, or secretion of molecules and signaling complexes. Trafficking of receptors, growth factors, nutrients, cytokines, integrins as well as pathogens dictates the kinetics and magnitude of signal transduction cascades. Understandably, alterations in the 'fate' of such cargo complexes have profound physiologic and pathophysiologic implications. Rab GTPases regulate endocytosis by decorating intracellular vesicles and targeting these vesicles along with their cargoes to appropriate subcellular compartments. In the last decade, the number of genetic diseases driven by germline mutations in Rab GTPases or their interacting proteins, has increased and there is growing evidence of aberrant Rab GTPase function in acquired pathophysiologies such as immune deficiency, infection, obesity, diabetes and cancer.     

Rab GTPases, intracellular traffic and disease.

Membrane and protein traffic in the secretory and endocytic pathways is mediated by vesicular transport. Recent studies of certain key regulators of vesicular transport, the Rab GTPases, have linked Rab dysfunction to human disease. Mutations in Rab27a result in Griscelli syndrome, caused by defects in melanosome transport in melanocytes and loss of cytotoxic killing activity in Tcells. Other genetic diseases are caused by partial dysfunction of multiple Rab proteins resulting from mutations in general regulators of Rab activity; Rab escort protein-1 (choroideremia), Rab geranylgeranyl transferase (Hermansky-Pudlak syndrome) and Rab GDP dissociation inhibitor-alpha (X-linked mental retardation). In infectious diseases caused by intracellular microorganisms, the function of endocytic Rabs is altered either as part of host defences or as part of survival strategy of the pathogen. The human genome is predicted to contain 60 RAB genes, suggesting that future work could reveal further links between Rab dysfunction and disease.     

Rab GTPases and myosin motors in organelle motility

The actin cytoskeleton is essential to ensure the proper location of, and communication between, intracellular organelles. Some actin-based myosin motors have been implicated in this process, particularly members of the class V myosins. We discuss here the emerging role of the Ras-like GTPases of the Rab family as regulators of myosin function in organelle transport. Evidence from yeast secretory vesicles and mitochondria, and mammalian melanosomes and endosomes suggests that Rab GTPases are crucial components of the myosin organelle receptor machinery. Better understood is the case of the melanosome where Rab27a recruits a specific effector called melanophilin, which in turn binds myosin Va. The presence of a linker protein between a Rab and a myosin may represent a general mechanism. We argue that Rabs are ideally suited to perform this role as they are exquisite organelle markers. Furthermore, the molecular switch property of Rabs may enable them to regulate the timing of the myosin association with the target organelle.     

Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway.

Synthetic peptides of the putative effector domain of members of the ras-related rab gene family of small GTP-binding proteins were synthesized and found to be potent inhibitors of endoplasmic reticulum (ER) to Golgi and intra-Golgi transport in vitro. Inhibition of transport by one of the effector domain peptides was rapid (t1/2 of 30 s), and irreversible. Analysis of the temporal site of peptide inhibition indicated that a late step in transport was blocked, coincident with a Ca2(+)-dependent prefusion step. The results provide novel biochemical evidence for the role of members of the rab gene family in vesicular transport in mammalian cells, and implicate a role for a new downstream Rab effector protein (REP) regulating vesicle fusion.     

Targeting Rab GTPases to distinct membrane compartments

Rab GTPases are key to membrane-trafficking events in eukaryotic cells, and human cells contain more than 60 Rab proteins that are localized to distinct compartments. The recent determination of the structure of a monoprenylated Rab GTPase bound to GDP-dissociation inhibitor provides new molecular details that are relevant to models of Rab delivery. The further discovery of an integral membrane protein that can dissociate prenylated Rab proteins from GDP-dissociation inhibitor gives new insights into the mechanisms of Rab localization.     

GRAB is a binding partner for the Rab11a and Rab11b GTPases

Co-ordination of Rab GTPase function has emerged as a crucial mechanism in the control of intracellular trafficking processes in eukaryotic cells. Here, we show that GRAB/Rab3IL1 [guanine nucleotide exchange factor for Rab3A; RAB3A interacting protein (rabin3)-like 1], a protein that has previously be shown to act as a GEF (guanine nucleotide exchange factor) for Rab3a, Rab8a and Rab8b, is also a binding partner for Rab11a and Rab11b, but not the closely related Rab25 GTPase. We demonstrate that exogenous expression of Rab11a and Rab11b shift GRAB’s distribution from the cytoplasm onto membranes. We find that the Rab11a/Rab11b-binding region of GRAB lies within its carboxy-terminus, a region distinct from its GEF domain and Rab3a-binding region. Finally, we describe a GRAB deletion mutant (GRAB_Δ223–228) that is deficient in Rab11-binding ability. These data identify GRAB as a dual Rab-binding protein that could potentially link Rab3 and Rab11 and/or Rab8 and Rab11-mediated intracellular trafficking processes.     

Association of Rab3A with synaptic vesicles at late stages of the secretory pathway

Rab3A is a small GTP-binding protein highly concentrated on synaptic vesicles. Like other small GTP-binding proteins it is thought to cycle between a soluble and a membrane-associated state. To determine at which stage of the life cycle of synaptic vesicles rab3A is associated with their membranes, the localization of the protein in neurons and neuroendocrine cells at different developmental and functional stages was investigated. In all cases, rab3A was colocalized with synaptic vesicle markers at the cell periphery, but was absent from the Golgi area, suggesting that rab3A associates with vesicles distally to the Golgi complex and dissociates from vesicle membranes before they recycle to this region. Immunofluorescence experiments carried out on frog motor end plates demonstrated that massive exocytosis of synaptic vesicles is accompanied by a translocation of rab3A to the cell surface. The selective localization of rab3A on synaptic vesicles at stages preceding their fusion with the plasmalemma suggests that the protein is part of a regulatory machinery that is assembled onto the vesicles in preparation for exocytosis.     

The diversity of Rab GTPases in Entamoeba histolytica

Rab proteins are ubiquitous small GTP-binding proteins that form a highly conserved family and regulate vesicular trafficking. Recent completion of the genome of the enteric protozoan parasite Entamoeba histolytica enabled us to identify an extremely large number (>90) of putative Rab genes. Multiple alignment and phylogenic analysis of amebic, human, and yeast Rab showed that only 22 amebic Rab proteins including EhRab1, EhRab2, EhRab5, EhRab7, EhRab8, EhRab11, and EhRab21 showed significant similarity to Rab from other organisms. The 69 remaining amebic Rab proteins showed only moderate similarity (<40% identity) to Rab proteins from other organisms. Approximately one-third of Rab proteins including Rab7, Rab11, and RabC form 15 subfamilies, which contain up to nine isoforms. Approximately 70% of amebic Rab genes contain single or multiple introns, and this proportion is significantly higher than that of common genes in this organism. Twenty-five Rabs possess an atypical carboxyl terminus such as CXXX, XCXX, XXCX, XXXC, and no cysteine. We propose annotation of amebic Rab genes and discuss biological significance of this extraordinary diversity of EhRab proteins in this organism.     

DENN domain proteins: regulators of Rab GTPases

The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways.     

Regulation of epithelial ion channels by Rab GTPases

Epithelial ion channels are crucial to many of life's processes and disruption of their functions can lead to several disorders. Cystic fibrosis, an autosomal recessive disorder, is caused by defects in the biosynthesis or function of the CFTR chloride channel. Similarly, mutations in certain ENaC genes leading to increased or reduced channel activity cause diseases such as Liddle's syndrome or PHA. In order for ion channel proteins to be functional they need to be expressed on the plasma membrane. Thus, molecules that modulate the trafficking of ion channels to and from the membrane are of utmost significance. Among the numerous factors that regulate their functioning is a family of small GTPases known as Rab proteins. While Rabs have always played a pivotal role in membrane trafficking, their diversity of functions and plethora of interacting partners have lately been brought to light. Recent studies reveal that multiple Rab isoforms physically interact with and/or modulate the activity of several ion channels. Rab proteins have the ability to serve as molecular switches and many of the ion channels are regulated differentially by the GTP- or GDP-bound Rab isoforms. This review examines the role of Rab GTPases in the trafficking of ion channels, including CFTR, ENaC, TRPV5/6, and aquaporins, based on recent evidence.     

Rab GTPases as regulators of transport through endosomes

Summary Early endocytic compartments are a highly dynamic, heterogeneous class of prelysosomal organelles that receive internalized proteins from the plasma membrane and sort these to various intracellular destinations. Several monomeric Rab GTPases are associated with the cytoplasmic surface of endosomes and regulate the dynamics of this endomembrane system. We discuss the endosomal Rab proteins and their effector proteins and how they might control vesicular transport through the endocytic pathway.