Rab GTPases and Membrane Trafficking

Finally, we review the mechanisms by which different Rabs communicate with one another in regulatory circuits that help to define each organelle and to establish the direction of membrane traffic. The intracellular localization of Rabs. A typical cell showing the intracellular localization and associated vesicle transport pathway s of several Rab GTPases.

Rab6 regulates intra-Golgi traffic. Several Rabs including Rab8, , and regulate biosynthetic traffic from the trans -Golgi network TGN to the plasma membrane. Several secretory vesicles and granules use Rab3, , , and to exocytose their cargo.

• Rab GTPases and Membrane Trafficking.
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Rab27 is well-studied in the melanosome transport that also relies on Rabs 32 and There are numerous Rabs associated with endosomal traffic, and the most active site of localization is the early endosome. Most early endocytic steps rely on Rab5, which mediates fusion of endocytic vesicles to form the early endosome. Traffic can be directed towards the lysosome for degradation, which relies on action of Rab7, or to various recycling compartments to return factors to the plasma membrane.

Rab15 directs membrane traffic from the early endosome to the recycling endosome.

Rab GTPases and Membrane Trafficking:: volume 1 | BenthamScience

Rab4 and Rab11 regulate fast and slow endocytic recycling, respectively. Specialized Rab functions include Rabmediated regulation of lipid droplets, intracellular lipid storage sites. Rab21 and Rab25 regulate transport of integrins to control cell adhesion and cytokinesis. Rab13 directs traffic to and regulates formation of tight junctions in polarized epithelial cells. Tight junctions define the boundary between the apical and basolateral regions of the polarized cell. Mutations in the mouse Rab23 gene lead to a severe developmental defect, open brain , because Rab23 acts downstream to negatively regulate Sonic hedgehog Shh signaling during dorsoventral development of the mouse spinal cord.

It potentially interacts with the transcription factors activated by the Shh pathway. Rab40 also acts in a signaling pathway; it recruits components of the ubiquitination machinery to regulate Wnt signaling. There are several poorly characterized Rabs, such as Rab It controls plasma membrane recycling of an essential factor in cytokinesis. Rab34 and Rab39 are found on the Golgi, but it is unclear what role they play. The Rab GTPase family: Rabs constitute the largest family of small Ras-like GTPases with 11 identified in yeast and more than 60 members in humans that can be classified in several phylogenetic and functional groups , The structures of at least 16 different Rab proteins in either their active GTP-bound or inactive GDP-bound state have been solved , Almost every group has at least one member represented as a crystal structure, allowing for some generalization regarding the specific structural features that contribute to Rab function COOH-terminal to the GTPase fold is the hypervariable region of the Rab followed by the CAAX boxes that normally contains two cysteine residues to which geranylgeranyl moieties are covalently attached.

These geranylgeranyl tails allow for regulated membrane insertion of the Rab that will be discussed in greater detail below. Because of the overall structural conservation, the differences between the active and inactive states must define the regions that determine the specific functions of each Rab. When GDP-bound, the switch regions tend to be disordered and undergo major changes to adopt a structurally well-ordered state upon binding GTP These structural differences explain how different Rab proteins recruit specific sets of effectors to regulate their respective pathways , , There are additional features of Rabs that contribute to their interactions with effector proteins and their mechanism of targeting to specific membranes.

A multiple sequence alignment of all known Rabs led to the identification of conserved stretches of amino acids, named F1—F5, that distinguishes Rabs from other members of the Ras superfamily The analysis also led to the identification of Rab subfamily-conserved sequences, named SF1— 4, that allowed for grouping of Rabs into various subfamilies and were predicted to define the sites of interactions with their respective effectors As the name implies, this portion of the Rab shows the greatest divergence in primary sequence among the different phylogenetic groups. This region has been shown to play a role in targeting of the Rab to specific membranes.

Replacement of the Rab5 hypervariable region with that of Rab7 targets the chimera to late endosomes that are normally marked by Rab7 In yeast, a similar chimera of Sec4 containing the hypervariable region of Ypt1 localized to Golgi structures that normally contain Ypt1 Chimeras of Rab1 or Rab5 with the hypervariable region of Rab9 that interacts with TIP47 can be relocated from the Golgi normal Rab1 localization or the early endosome normal Rab5 localization to the late endosome normal Rab9 localization upon overexpression of TIP47 1. More recent studies in mammalian cells show that certain F and SF regions of Rabs are more important than their hypervariable domains for membrane targeting and implicate interactions with effector proteins for proper localization 3 , It is important to note that the hypervariable region contains a motif that interacts with proteins that regulate the membrane-bound state of the Rab see below.

Therefore, the conflict in targeting mechanisms may reflect the different pathways being studied and the overall contribution of multiple Rab motifs and interacting partners to membrane localization. Rab proteins cycle between the cytosol and the membrane of its respective transport compartment Fig. The nucleotide-bound state of the Rab influences its localization and activity Fig. The active Rab now interacts with effector proteins that specifically facilitate traffic in its respective pathway.

The Rab, bound to GDI, is now ready to be reinserted into a membrane and begin the cycle again. REP delivers the Rab to its target membrane. Throughout this process, the Rab is GDP-bound. The GTP-bound Rab interacts with effector proteins that mediate membrane traffic in the pathway regulated by its associated Rab. The Rab is then removed from the membrane by guanine nucleotide dissociation inhibitor GDI in preparation for the next cycle.

Loss-of-function mutations at each of the above steps produce disease phenotypes as indicated by the red text boxes. The Rab cycle is critical for regulating traffic to and from particular organelles and thus helps to define their identity. Any perturbation in the steps described above can result in a variety of disease states Fig.

Mutations in the human REP-1 gene lead to choroideremia, a disease characterized by progressive atrophy of the choroid, retinal pigment epithelium, and retina that lead to eventual blindness The cause of the disease is most likely due to loss of Rab27A function, which accumulates in an unprenylated form in retinal tissue samples from patients with the disease. Modulation of RabGGT function has also been shown to play a role in several diseases. Bisphosphonate drugs that inhibit geranylgeranylation of Rab proteins have been used to remedy bone diseases characterized by excessive resorption, such as osteoporosis 88 , These drugs have also been shown to induce apoptosis in certain types of cancers These data correlate well with the identification of several Rab proteins as cancer markers.

This is discussed in detail below in this article. Mutations in the human GDI1 gene lead to X-linked nonspecific mental retardation Mice carrying a deletion of the Gdi1 gene have defects in short-term memory formation and social interaction patterns that is phenotypically similar to humans carrying GDI1 mutations Analysis of brain extracts from mutant mice revealed an accumulation of membrane-bound Rab proteins, but Rab4 and Rab5, both of which regulate endosomal traffic, were more significantly affected than other Rab proteins analyzed Mutations in the genes encoding the regulatory and catalytic subunits of the Rab3GAP lead to Warburg Micro and Martsolf syndromes, diseases characterized by developmental abnormalities of the eye, nervous system, and genitalia 4 , 5.

Rab3A is the most abundant Rab found in the brain and regulates exocytosis of synaptic vesicles , , A Rab GEF has also been implicated in human disease. Mutations in the human SEDL gene, the homolog of the yeast TRAPP subunit Trs20, lead to spon-dyloepiphyseal dysplasia tarda, an X-linked disorder characterized by disproportionately short stature, a short neck and trunk, and degeneration of the spine and hips , , , , These are clear examples of physiological disorders that arise from disrupting the Rab cycle.

Although Rabs in general are strikingly similar in their overall structure, the proteins that interact with them, to either regulate their activity or carry out their downstream functions are not. Recent crystal structures illustrate several distinct mechanisms by which GAPs and GEFs regulate the nucleotide-bound state of Rab proteins. The structures of GDI and REP cocrystallized with Rabs show how they associate with Rab proteins and their hydrophobic geranylgeranyl tails that mediate membrane insertion.

Several features distinguish the functions of GDI and REP and thereby allow them to play different roles in the life cycle of a Rab protein. They are structurally similar and related in function by their affinity for the GDP-bound form of Rabs and their ability to interact with the Rab geranylgeranyl tails. However, REP binds with high affinity to the GDP-bound Rab protein either prenylated or unprenylated, while GDI binds tightly to the Rab with its prenyl groups and binds poorly to the unprenylated Rab protein , The structures of GDI bound to mono- and di-geranylgeranylated Ypt1 and REP bound to mono-geranylated Rab7 help to distinguish their functions 7 , 11 , , , — The structures show strong conservation in their interaction with the switch and interswitch domains of their associated Rab, maintaining it in a GDP-bound state.

Domain I also contains the binding site for the aliphatic-X polar -aliphatic AXA motif in the Rab hypervariable region while in domain 2, both geranylgeranyl motifs bind in the same prenyl-binding pocket. The more constricted prenyl-binding pocket of REP compared with GDI suggests the second prenyl group may bind outside of the pocket and partially displace the first geranylgeranyl moiety to reduce its overall affinity for REP.

The higher affinity for monoprenylated Rab7 may ensure a second geranylgeranyl group is attached to the Rab as Rabs with only one prenyl group tend to be retained at the ER and do not move to their normal intracellular location In the case of GDI, it binds poorly to unprenylated Rabs but with high affinity to mono- and diprenylated Rab7. The structure of GDI with di-geranylgeranylated Ypt1 shows both groups in an overlapped arrangement in the prenyl-binding pocket This implies that the opposite reaction would require additional factors, such as a GDF or the molecular chaperone Hsp90, to efficiently break the stable Rab-GDI interaction 67 , , , The GDF Yip3, an integral membrane protein found on endosomes, has been shown to catalyze dissociation of GDI from Rab9 through an as yet uncharacterized mechanism , The opposing GDI-mediated Rab extraction and GDF-mediated Rab insertion mechanisms are undoubtedly related, and uncovering one mechanism will likely shed light on the other.

The crystal structure of the TBC domain of Gyp1, the GAP for Ypt1, revealed the mechanism to be dependent on a conserved arginine finger that interfaces with the Rab nucleotide binding pocket to stimulate GTP hydrolysis , On the basis of the crystal structure, the fundamental GAP mechanism of Gyp1 was expected to be the same as that of GAP proteins for Ras or Cdc42, despite significant overall structural differences.

This GAP mechanism is likely to be conserved among all Rab-GAP combinations, but additional structures will be needed to test this prediction. However, the structures of several GEF proteins indicate that they directly insert into, or indirectly alter, the Rab nucleotide or magnesium-binding site to cause displacement of the bound nucleotide The crystal structure of Sec2-Sec4 complex was recently solved and revealed the mechanism by which the coiled coil Sec2 dimer facilitates nucleotide exchange on the Rab Sec4 Sec2 interacts with residues in the switch I and switch II domains of Sec4 to induce structural changes in the nucleotide binding pocket that reduce its affinity for nucleotide.

No part of Sec2 directly inserts into the nucleotide binding pocket of Sec4, unlike the Rabex5 mechanism of nucleotide exchange Sec4 is the closest yeast homolog of Rab8, and Rabin8 is a GEF for Rab8 that shares a region of homology with the catalytic site of Sec2p Sec4 and Rab8 interact with two other related proteins, Dss4 and Mss4, respectively. These are much less efficient than Sec2 and Rabin8 in catalyzing exchange , and in the case of Dss4, it only stimulates dissociation of GDP, not the subsequent binding of GTP Mss4 has also been shown to form a stable association with other Rab proteins on both exocytic and endocytic pathways, and this activity of Mss4 may relate to its proposed function as a general chaperone for misfolded Rab proteins rather than a specific GEF 47 — 49 , , , The crystallized complex contains two copies of Bet3 and one copy each of Bet5, Trs23, and Trs Rab proteins regulate their respective pathways by interacting with various effector proteins.

Effectors are generally defined as proteins that preferentially interact with the GTP-bound form of their respective Rab, although there are examples, such as protrudin, that interact preferentially with the GDP-bound form of Rab11 Different Rab effectors act during vesicle formation, movement, tethering, and fusion, with each pathway having its own unique set of effectors Fig. We begin by highlighting some of the best-characterized Rab effectors and their specific functions in membrane traffic.

Rabs perform their regulatory function by recruiting a variety of effectors to mediate different functions in membrane transport. These functions are as follows: Below each function are examples of Rab effectors that perform said function. Mutations in Rab effectors also lead to disease phenotypes: Vesicle cargo selection is determined by components of each coat complex that recognize specific elements of the cargo to be transported. However, several Rab proteins also have been shown to participate in this process.

The best example of this involves Rab9, which regulates membrane traffic between late endosomes and the trans -Golgi network TGN TIP47 is a Rab9 effector that interacts with the cytoplasmic domain of mannosephosphate receptors and is required for them to be recycled from endosomes to the TGN 1 , 60 , The interaction of Rab9 with TIP47 enhances the interaction between the mannosephosphate receptor and TIP47 during the formation of the transport vesicle. Another example of a complex that acts to appropriately select cargo is the retromer complex.

It is required for retrieval of transmembrane proteins from endosomes to the TGN 36 , The SNXs contain a PX phox homology domain that interacts with phosphoinositides and a BAR domain that can serve as a multimerization interface to induce membrane curvature 58 , The VpsVpsVps35 trimer is responsible for cargo binding, and the sequential actions of Rab5 and Rab7 are required for recruiting this trimer complex Rab5 is important for phosphoino-sitide regulation through its effector, phosphatidylinositol 3-kinase, while the retromer trimer is an effector of Rab7.

It is not known if Rab7 influences the interaction of retromer with cargo. In addition to selecting cargo, Rab proteins recruit effectors that are critical for vesicle movement along actin- or microtubule-based cytoskeletal structures. There are several outstanding examples of such effectors. Rab11 in mammalian cells interacts with myosin Vb through its effector, Rab11 family interacting protein 2 RabFIP2 , to regulate plasma membrane recycling This tripartite complex is physiologically important because mutations in any one member lead to the rare autosomal recessive disorder Griscelli syndrome GS , first identified by the mouse mutants dilute, leaden, and ashen Myo5a, Rab27a, and melanophilin, respectively These patients display a range of symptoms from hypopigmentation GS3, melanophilin mutation to immunological defects GS2, Rab27a mutations and neurological impairments GS1, MyoVa mutations.

Rab proteins are also involved in movement of organelles. Yeast cells utilize different pathways, some of which share factors in common, to ensure that the daughter cell acquires the full complement of organelles necessary for survival. Organelles generally utilize the actin cytoskeleton, appropriately polarized through the action of formin proteins, as a track for transport by a type V myosin from the mother cell to the bud The Rab Ypt11 has been shown to regulate the inheritance of both mitochondria and Golgi in yeast by recruiting the type V myosin Myo2 as an effector 17 , 34 , Although Golgi appear to travel by associating with Myo2, mitochondrial movement may be powered in part by actin polymerization, and the recruitment of Myo2 by Ypt11 is necessary for retaining mitochondria at the poles of mother and daughter cell during the cell cycle In animal cells, many membrane traffic pathways rely on microtubules, and Rabs have been shown to interact with microtubule-based motors to regulate these pathways.

Microtubules are generally organized with their minus ends at microtubule organizing centers, such as the centrosome, and direct their plus ends into the cytoplasm and towards the cell periphery. Rab proteins can regulate traffic in either direction by interacting with members of the kinesin plus-end directed motors or dynein minus-end directed motors family. Dynein is normally in a complex with dynactin that couples the motor to and stimulates vesicle motility along microtubules , , , Rab6 localizes to the Golgi and primarily regulates retrograde traffic between endosomes, Golgi, and the ER but has recently been shown to also be involved in exocytic traffic to the plasma membrane 99 , , , , , , , , , Rab6 interacts directly with Rabkinesin-6 kinesin family member 20A to facilitate intra-Golgi transport , Rab7, which coordinates traffic between late endosomes and the lysosome, interacts with Rab-interacting lysosomal protein RILP to recruit the dynein-dynactin motor complex to transport late endosomes towards centrosomes and the lysosome , This particular Rab-effector interaction is of interest because it is manipulated by several intracellular pathogens.

The Salmonella SifA protein prevents the recruitment of RILP by Rab7 to facilitate growth of the membrane-bound compartment in which the bacterium can replicate , Heliobacter pylori takes advantage of this interaction to create a bacterium-containing vacuolar compartment that requires Rab7 and RILP to direct endosomal traffic to it , Most membrane traffic pathways utilize coated vesicles of one sort or another, and these coats must be shed to allow the vesicles to fuse with their target membrane.

In addition to playing a role in coat formation, Rabs may also play a role in uncoating. Rab5 regulates the early endocytic pathway and is found on clathrin-coated vesicles CCVs. Recruitment of clathrin to newly forming endocytic vesicles is primarily through the assembly polypeptide 2 AP-2 clathrin adaptor complex that recognizes and binds to both cargo i.

Phosphatidylinositol-4,5-bisphosphate [PI 4,5 P 2 ], a phosphoinositide that is normally found at the plasma membrane, is also a significant component for recruiting AP-2 during clathrin-mediated endocytosis , Rab5 regulates CCV uncoating in two ways: Ypt1, or Rab1, is required for ER-to-Golgi traffic and presumably recruits factors that facilitate uncoating of COPII vesicles in preparation for fusion , , , , These tethering factors fall into two categories: Members of both categories of tethers are Rab effectors, and some also regulate the nucleotide-bound state of their associated Rabs such as the TRAPP complex described above.

Despite differences in structure and organization, all of these tethering factors ensure fidelity in transport as they regulate SNARE-mediated fusion of their respective vesicles to the target membrane.

I. INTRODUCTION

The Golgins are a family of coiled-coil tether proteins with members that include p Uso1 in yeast , giantin, and GM Sequence analysis suggested an evolutionary relationship of p with Uso1, a protein previously defined as an essential factor in ER to Golgi transport in yeast , , , Both Uso1 and p are homodimers that consist of a long coiled-coil tail that binds to factors such as the cis -Golgi-localized GM and the COPI vesicle factor giantin and a globular head that binds to Rab1 6, 10, , , , a.

The GRIP domain mediates an interaction with the Arf-like protein Arl1 to participate in trans -Golgi recruitment of the golgin , , unlike the above golgins normally found at the cis- Golgi. These golgins would therefore potentially serve as scaffolds to recruit traffic from multiple Rab-regulated pathways to the correct side of the Golgi.

Although the significant players in the process have been identified, defining how they interact at a molecular level to regulate ER-to-Golgi and intra-Golgi traffic still requires more work. Another coiled-coil tether protein is early endosome antigen 1 EEA1 , an effector of Rab5 that is involved in tethering and fusion of early endosomes , , As a dimer, EEA1 is thought to bridge endosomes through its FYVE domain, an evolutionarily conserved phosphati-dylinositol 3-phosphate [PI 3 P] binding motif, and through its interaction with the SNARE protein syntaxin 6 to mediate homotypic endosomal fusion 55 , 56 , , , , Therefore, similar to Rab1, Rab5 interacts with coiled-coil tethers to connect membranes and specific SNARE proteins that mediate fusion in their respective pathways.

In most cases, vesicle tethering is performed by multisubunit complexes. There are eight known complexes: From recent structural data, an emerging theme is the structural similarity of several tethering complexes and their interface with components of the SNARE machinery as a mechanism of regulating fusion. A recent discovery proved insightful: The exocyst is an octameric complex that tethers secretory vesicles to the plasma membrane in preparation for fusion , The vesicle-associated Rab Sec4 recruits the exocyst by interacting with one of its subunits, Sec15, as an effector protein The exocyst is unique in that some of its subunits are also effectors of Rho proteins found on the plasma membrane.

This arrangement presumably ensures efficient and accurate tethering to sites marked by these polarity determinants , , Furthermore, the exocyst has both direct and indirect interactions with components of the SNARE machinery. It is unclear how exactly Sec4, Rho proteins, and SNAREs interact with the exocyst to control the fusion of secretory vesicles at the plasma membrane. Exocyst subunits, as rods, can potentially bundle together in a side-by-side fashion and perhaps in parallel to the two opposed vesicular and plasma membranes. This would bring together the SNARE proteins found on the opposing membranes, as well as Sro7 and Sec1 to regulate their assembly and function, and this process is controlled by the concurrent interactions of several exocyst subunits with Sec4 on vesicles and Rho proteins at the plasma membrane The conserved oligomeric Golgi COG complex is composed of eight subunits and regulates retrograde traffic within the Golgi as well as between the endosome and the Golgi COG plays a role in recycling Golgi resident proteins, highlighted by the observation that mutations in subunits of the COG complex produce defects in glycosylation that lead to severe congenital disease phenotypes , In addition to being structurally similar to the COG2 subunit, COG4 is remarkably similar in structure to Sec6 as well as the other solved structures of exocyst proteins This observation is discussed further below.

The structure of Vps54 revealed that the mutation responsible for the wobbler mouse phenotype, which leads to spinal muscular atrophy and serves as an animal model for amyotrophic lateral sclerosis, destabilizes Vps54 and results in reduced levels of the protein and of the GARP complex A potential link may be Rab6 interacting protein 1, a protein that interacts with Rab6, Rab11, and the retromer , No known Rab has been shown to participate in this process.

How these common structural features contribute to the tethering process and SNARE function are undoubtedly a critical focus of future research. The core of both complexes is composed of the class C Vps proteins Vps11, Vps18, Vps16, and Vps33 , first identified in yeast through the isolation of mutants that produce no identifiable vacuoles 22 , 23 , , , The HOPS complex and Ypt7 are required for efficient and accurate homotypic fusion of vacuolar membranes 79 , , , , , Both Vps3 and Vps8 are members of the Vps class D proteins, identified through the isolation of mutants with enlarged vacuoles, and are implicated in sorting of proteins to endosomes 71 , In addition to recruiting tethers that ensure the proper association of cargo and target membranes, Rab proteins also regulate the SNARE-dependent fusion of transport and target membranes.

The Rab Sec4 is a yeast homolog of Rab8 and regulates the final stage of the secretory pathway in yeast. A recently discovered effector of Sec4 is Sro7, a member of the lethal giant larvae lgl family of proteins that interacts with Sec9 and regulates SNARE function Several mutations that disrupt the secretory pathway can be rescued by overexpression of Sec4, and this mechanism of rescue requires the function of Sro7 , Rab5 is found on early endosomes and plays a critical role in targeting endosomal traffic towards lysosomes through the function of its numerous effectors.

Recruitment of effectors using this dual mechanism is physiologically important because in the absence of Vps34 function, recruitment of both EEA1 and rabenosyn-5 is prevented and fusion of early endosomes is blocked , , , As membrane flows from one organelle to another, it must transition through different Rab-defined compartments.

To what extent the compartment defines the Rab, or vice versa, has been an open question, which has been framed primarily by studies of specific pathways, the Rab proteins that are involved, and how they are each activated and inactivated to generate a programmed transition from one Rab to the next. How does this process occur? What mechanisms ensure the directionality of the switch and that the compartment is ready to receive the next Rab and its set of effectors?

In several specific cases, recent evidence supports a maturation model whereby the compartment transitions from an upstream Rab to a downstream Rab by recruiting as effectors the GAP and GEF for the upstream and downstream Rabs, respectively Fig. The countercurrent cascades of GAPs and GEFs not only ensure that the appropriate downstream Rab is recruited but that the upstream Rab is concomitantly inactivated to delineate one compartment from another.

1. Rab GTPases and Membrane Trafficking.
2. UN TROP BEAU REVE (French Edition);
3. Reality Today Forum (February 2012).
4. Role of Rab GTPases in Membrane Traffic and Cell Physiology.
5. Grandmas Garden.

GTP-bound RabB performs two functions: The concomitant action of GAPs and GEFs ensures the boundaries of each membrane compartment, determined by the actions of their associated Rab, are well-defined. By activating Sec4, Sec2 promotes the stable association of Sec4 with secretory vesicles that ensures their delivery to and fusion with the plasma membrane In this manner, Sec4 is assured of its involvement in the correct pathway because its association with secretory vesicles is dependent on the Rab directly upstream of it.

Furthermore, Sec2 also associates with Sec15, a component of the exocyst and an effector of Sec4 as an additional mechanism to recruit Sec4 to secretory vesicles , , Rabex5 interacts with Rabaptin5, an effector of Rab5, to ensure proper spatiotemporal activation of Rab5 A somewhat related example of an effector playing a role in targeting a Rab to a specific membrane involves the interaction of Rab9 with its effector TIP Several chimeras of Rab1 or Rab5 with the hypervariable region of Rab9 that interacts with TIP47 can be relocated from the Golgi normal Rab1 localization or the early endosome normal Rab5 localization to the late endosome normal Rab9 localization upon overexpression of TIP47 1.

This result indicates the importance of the Rab-effector interaction in determining the proper localization of the Rab of interest. Endocytic cargo is initially found in Rab5-containing early endosomal compartments that can undergo maturation to become Rab7-containing late endosomal compartments targeted for lysosomes This process of Rab conversion has been visualized in mammalian cells and appears to progress in several steps: These data suggest a maturation model whereby each transport compartment gains the necessary factors to move forward along the pathway while losing those that define the previous compartment Additional support for the maturation model comes from elegant studies of the Golgi in Saccharomyces cerevisiae.

Both studies show specific Golgi cisternae transitioning from being marked with an early Golgi marker to a late Golgi marker at a rate consistent with that seen for cargo transitioning through the secretory pathway , In the early endocytic pathway, the early endosome serves as a hub for traffic directed in several different directions through the action of various Rabs. Another Rab5 effector, rabenosyn-5, has a binding site for and is an effector of Rab4 that is involved in targeting proteins to the Rabpositive recycling endosome Overexpression of rabenosyn-5 leads to prolonged overlap of Rab5 and Rab4 and shows how a divalent effector can influence Rab conversion and target traffic appropriately from a compartment that serves multiple pathways.

The GEF cascades above describe how a Rab conversion can be initiated. However, to avoid an extended period of overlap of Rab domains within a compartment, it is also important to inactivate the upstream Rab once the downstream Rab has been recruited and activated. GAPs are the primary players in this process. In yeast, compartments marked with Ypt1 at the early Golgi mature to contain Ypt32 at the late Golgi. A key step in this process is the recruitment of Gyp1, the GAP for Ypt1, by Ypt32 to inactivate Ypt1 and promote its removal from membranes Loss of Gyp1, which is normally found at the Golgi as an effector of Ypt32, results in the prolonged overlap of Ypt1 and Ypt32 in a Golgi compartment.

However, several Rabs have also been implicated in the progression of multiple cancers 75 as membrane traffic plays a significant role in cancer biology, primarily in the loss of cell polarity and in the metastatic transformation of tumor cells This includes the upregulation of Rab5 in malignant and met-static human lung cell adenocarcinoma, Rab1 in tongue squamous cell carcinomas, and Rab3 in cancers of the nervous system Rab5 is an appropriate target due to its role in receptor-mediated endocytosis. Modulating Rab5 function can significantly alter signaling from growth factors to promote tumorigenesis, and both up-and downregulation of Rab5a is associated with cancer in different tissues 90 , , The best characterized example of a Rab implicated in cancer is Rab25, a Rab closely related to Rab11 that regulates apical endocytosis and transcytosis in epithelial cells 61 , , Rab25 is upregulated in certain ovarian and breast cancers due to amplification of a chromosomal region containing the Rab25 gene.

The resulting overexpression of Rab25 is associated with more aggressive forms of the associated cancer and a lower patient survival rate Therefore, Rab25 does not play a role in tumor initiation but facilitates its progression by allowing it to be invasive. Recent discoveries implicate Rabs in several prevalent neurological diseases. Neurons may be more sensitive to perturbations in membrane traffic because of their unique polarized structure and function.

Specialized functions of Rabs are critically important for synaptic function Rab3 , neurite growth and remodeling Rab11 and Rab13 , and general nervous system development Rab23 The resulting mutation produces an NH 2 -terminal polyglutamine repeat, and the length of the expansion, and subsequently the polyQ repeat, correlates inversely with age of onset , It is unclear how the polyQ repeat produces a disease state, but htt is normally associated with membranes and plays a role in membrane traffic , A recent study shows that mutant htt disrupts clathrin-dependent post-Golgi traffic targeted for lysosomes Mutant htt prevents the association of Rab8 with optineurin at the Golgi and results in reduced AP and clathrin-mediated traffic to lysosomes.

Htt interacts with the optineurin protein and FIP-2 that are both effectors of Rab8 at the Golgi , , Rab8 and FIP-2 recruit htt to the Golgi, and the interaction of optineurin with myosin VI is important for maintaining Golgi structure However, it is not known what role htt normally plays in its association with Rab8, FIP-2, and optineurin at the Golgi. Membrane fractions from mutant mouse brains did not catalyze nucleotide exchange on Rab11, and a Rab11 dominant-negative mutant expressed in normal adult brains led to neurodegeneration similar to the HD mutant mouse model. Very recent data show delayed recycling of transferrin to the plasma membrane and impaired Rabdependent vesicle formation from recycling endosomes in fibroblasts from Huntington patients compared with healthy individuals Both Rab8 and Rab11 localize to the recycling endosome RE and target proteins for the plasma membrane, although it is unclear how the two Rabs are differentiated in function 15 , 16 , , It will be interesting to see how the interplay between Rab8, Rab11, and htt become altered during the onset of HD and how that contributes to the pathophysiology of the disease.

Carpenter syndrome is an autosomal recessive disorder with symptoms that include skull abnormalities, poly-dactyly, brachydactyly shortness of fingers and toes , obesity, congenital heart disease, and mental retardation Mutations in Rab23 have been identified as the causative agent of the disease, and surprisingly, the associated phenotypes differ quite dramatically from the mouse Rab23 open brain opb mutant that is embryonically lethal , , Rab23 acts as a negative regulator of sonic hedgehog shh signaling during dorsal-ventral axis formation of the neural tube.

By activating Rab23, dorsal neural cells can prevent shh signaling that is required for ventral cells of the spinal cord Rab23 signaling through shh is more than likely the cause of symptoms seen in Carpenter syndrome as mutations in shh signaling components also produce phenotypes such as polydactyly and brachydactyly However, Rab23 was first cloned as a Rab predominantly expressed in the mouse brain , and although there are potential similarities, Carpenter syndrome phenotypes are more pleiotropic than those seen for opb mice The Rab23 mutations that cause Carpenter syndrome may have uncovered novel signaling pathways involving shh that will require further attention to characterize these connections.

Rab7 is a critical regulatory component that directs traffic in the endosomal pathway to the lysosome 44 , Point mutations in Rab7 lead to Charcot-Marie-Tooth disease type 2B, an inherited motor and sensory neurological disorder characterized primarily by distal muscle weakness and atrophy 24 , , , Biochemical analysis indicates that Rab7 carrying any of the identified point mutations is preferentially GTP bound and has a slower rate of GTP hydrolysis 96 , It is interesting to note that mutations in an endocytosis-related gene, dynamin 2, that impair clathrin-mediated endocytosis also produce Charcot-Marie-Tooth disease phenotypes 33 , , The above examples show how Rab-regulated pathways can be perturbed to cause disease.

In a related manner, Rabs and their effectors have become targets for infectious microorganisms that have developed mechanisms to evade host defenses by hiding and replicating in an intracellular environment. To avoid the host cell degradation machinery and obtain nutrients and building blocks to multiply, such organisms manipulate several different Rabs to their advantage. Salmonella enterica and Serovar typhimurium , the cause of gastroenteritis commonly referred to as salmonellosis, are initially taken up by epithelial cells that line the gut.

They reside in Salmonella -containing vacuoles SCVs in the cell that transition from a Rab5- to a Rab7-containing compartment 21 , , , , Rab7 effectors position the compartment at a perinuclear location close to the Golgi 39 , Acidification of the compartment causes release of Salmonella virulence factors that act to block the compartment from fusing with the lysosome, anchor the SCV to the Golgi, and recruit traffic from the Golgi 21 , 43 , , For example, the Salmonella SopB protein, a PI phosphatase, recruits sorting nexin 1 Snx1 to the SCV membrane for retromer-mediated removal of mannosephosphate receptors from its membrane 46 , The SopB protein, therefore, prevents maturation by enhancing recycling of unwanted lysosomal proteins from the SCV.

The SCVs accumulate a variety of Rab proteins on their membranes but not those indicative of phagosomes undergoing a normal maturation process towards lysosomes It is unclear how SCVs bypass this process. A key component is the Chlamydia Inc protein Cpn that has similar features to Golgin proteins and interacts with Rab1, Rab10 and Rab11 The volume of information describing Rab function in membrane traffic has grown dramatically in recent years.

In addition to identifying the many Rab proteins, defining their subcellular localizations, and isolating their regulators and effectors, we are beginning to understand how Rabs communicate with each other to specify where their respective territories begin and end. Although we have provided a few examples of how one Rab domain might transition to another, it is presently unclear if these mechanisms are universally applicable to all Rab-regulated pathways.

If not, how do these other Rabs determine the pathways that they regulate? However, do GDFs also play a role in this process? Does each pathway have a specific GDF, or are they shared among sets of pathways? How is this sharing regulated? We may have identified the major factors that regulate Rab function, but establishing how they are coordinated to achieve a common goal will require further analysis. While several Rabs have been very intensively studied, a large fraction of the Rab proteins expressed in mammalian cells have not, and relatively little is known regarding their function and regulation.

A recent study indicated that 42 Rab GTPases are expressed in COS7 cells, with the abundant Rabs being those that regulate endocytosis, secretion, and traffic to, from, and within the Golgi Are these uncharacterized Rabs simply redundant with the better-known members of their branch of the Rab family or have they acquired unique functions?

Do these Rabs serve tissue-specific roles? Will the same mechanisms act to control their function? How do they interact with the other Rabs found inside the cell? To understand the forces underlying the dramatic expansion of the Rab family during evolution, we must begin by describing the function of each Rab in greater detail.

Knockouts and knockdowns of the less-studied Rabs, both singly and in a combinatorial fashion, will help to reveal the common and unique functions of each Rab. In vitro assays using donor and target membranes and all identified factors are now a realistic goal for many Rabs. In addition to describing Rab function at a molecular level, assays such as these can be used to identify and analyze novel factors that affect the pathway of interest.

Rabs are involved in the pathogenesis of a wide range of diseases but exactly what role they play in some of these disorders is still unclear. Recent discoveries of the interaction of Rab35 with the actin bundling protein fascin to regulate intracellular actin assembly or the function of Rab23 in brain and chondrocyte development highlight the diverse roles of Rab proteins. Their involvement in signaling pathways outside of their stereotypical role in membrane traffic only magnifies our need to investigate in greater detail how Rabs work.

No conflicts of interest, financial or otherwise, are declared by the author s. National Center for Biotechnology Information , U. Author manuscript; available in PMC Jul Author information Copyright and License information Disclaimer. Address for reprint requests and other correspondence: The publisher's final edited version of this article is available free at Physiol Rev. See other articles in PMC that cite the published article. Abstract Intracellular membrane traffic defines a complex network of pathways that connects many of the membrane-bound organelles of eukaryotic cells.

Open in a separate window. Rab Proteins and Vesicle Movement In addition to selecting cargo, Rab proteins recruit effectors that are critical for vesicle movement along actin- or microtubule-based cytoskeletal structures. Rab Proteins and Vesicle Uncoating Most membrane traffic pathways utilize coated vesicles of one sort or another, and these coats must be shed to allow the vesicles to fuse with their target membrane. Coiled-coil tethers The Golgins are a family of coiled-coil tether proteins with members that include p Uso1 in yeast , giantin, and GM Multisubunit tethers In most cases, vesicle tethering is performed by multisubunit complexes.

The exocyst The exocyst is an octameric complex that tethers secretory vesicles to the plasma membrane in preparation for fusion , The COG complex The conserved oligomeric Golgi COG complex is composed of eight subunits and regulates retrograde traffic within the Golgi as well as between the endosome and the Golgi Rabs and Membrane Fusion In addition to recruiting tethers that ensure the proper association of cargo and target membranes, Rab proteins also regulate the SNARE-dependent fusion of transport and target membranes.

Sec4 and Sro7 The Rab Sec4 is a yeast homolog of Rab8 and regulates the final stage of the secretory pathway in yeast. Rab5 interacts with rabenosyn-5 and EEA1 Rab5 is found on early endosomes and plays a critical role in targeting endosomal traffic towards lysosomes through the function of its numerous effectors. From Rab5 to Rab7 Endocytic cargo is initially found in Rab5-containing early endosomal compartments that can undergo maturation to become Rab7-containing late endosomal compartments targeted for lysosomes Carpenter Syndrome and Rab23, Charcot-Marie-Tooth Disease and Rab7 Carpenter syndrome is an autosomal recessive disorder with symptoms that include skull abnormalities, poly-dactyly, brachydactyly shortness of fingers and toes , obesity, congenital heart disease, and mental retardation Salmonella enterica and Chlamydia pneumonia Salmonella enterica and Serovar typhimurium , the cause of gastroenteritis commonly referred to as salmonellosis, are initially taken up by epithelial cells that line the gut.

TIP47 is a key effector for Rab9 localization. Rab escort protein-1 is a multifunctional protein that accompanies newly prenylated rab proteins to their target membranes. Ali B, Seabra M. Targeting of Rab GTPases to cellular membranes. Am J Hum Genet. Alory C, Balch W. Rab32 is an A-kinase anchoring protein and participates in mitochondrial dynamics. Characterization of human Rab20 overexpressed in exocrine pancreatic carcinoma.

Structural and functional analysis of the globular head domain of p provides insight into membrane tethering. The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmic reticulum retrieval in yeast. Andag U, Schmitt H. Dsl1p, an essential component of the Golgi-endoplasmic reticulum retrieval system in yeast, uses the same sequence motif to interact with different subunits of the COPI vesicle coat. Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells.

Ypt11 functions in bud-directed transport of the Golgi by linking Myo2 to the coatomer subunit Ret2. Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells. The GTP-binding protein Ypt1 is required for transport in vitro: Isolation of yeast mutants defective in protein targeting to the vacuole. Organelle assembly in yeast: A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins. Identification and characterization of Iporin as a novel interaction partner for rab1.

A cryptic Rab1-binding site in the p tethering protein. RAB39A binds caspase-1 and is required for caspasedependent interleukin-1beta secretion. Benmerah A, Lamaze C. The small GTPase Rab4A interacts with the central region of cytoplasmic dynein light intermediate chain Biochem Biophys Res Commun. Dynamin 2 mutations associated with human diseases impair clath-rin-mediated receptor endocytosis.

Role of Rab GTPases in Membrane Traffic and Cell Physiology

A type V myosin Myo2p and a Rab-like G-protein Ypt11p are required for retention of newly inherited mitochondria in yeast cells during cell division. Insights into the biogenesis of lysosome-related organelles from the study of the Hermansky-Pudlak syndrome. Ann NY Acad Sci. Bonifacino J, Hurley J. Curr Opin Cell Biol. Bonifacino J, Rojas R. Retrograde transport from endosomes to the trans -Golgi network. Nat Rev Mol Cell Biol. The intra-cellular fate of Salmonella depends on the recruitment of kinesin. Brennwald P, Novick P. Brumell J, Scidmore M. Manipulation of rab GTPase function by intracellular bacterial pathogens.

Microbiol Mol Biol Rev. Eur J Cell Biol. Sorting nexin-1 defines an early phase of Salmonella -containing vacuole-remodeling during Salmonella infection. Burton J, De Camilli P. A novel mammalian guanine nucleotide exchange factor GEF specific for rab proteins. Adv Second Messenger Phosphoprotein Res. A mammalian guanine-nucleotide-releasing protein enhances function of yeast secretory protein Sec4. Mutants in trs disrupt traffic from the early endosome to the late Golgi. Direct interaction of EEA1 with Rab5b. The endosome fusion regulator early-endosomal autoantigen 1 EEA1 is a dimer.

Rab-interacting lysosomal protein RILP: Sorting nexins—unifying trends and new perspectives. Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells. Ypt32p and Mlc1p bind within the vesicle binding region of the class V myosin Myo2p globular tail domain. Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3D microenvironments.

Structural analysis of conserved oligomeric Golgi complex subunit 2. Hypervariable C-terminal domain of rab proteins acts as a targeting signal. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Chen C, Balch W. Rab36 regulates the spatial distribution of late endosomes and lysosomes through a similar mechanism to Rab Rab39, a novel Golgi-associated Rab GTPase from human dendritic cells involved in cellular endocytosis.

Chen Y, Stevens T. The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic beta-cell exocytosis. Chia W, Tang B. Emerging roles for Rab family GTPases in human cancer. What's new in Forthcoming Titles in Forthcoming Series. End User License Agreement. Table of Contents Foreword - Pp. List of Contributors - Pp.

An Overview - Pp. Rab6 GTPase - Pp. Polarized Exocytosis in Yeast: Masterclass with Rab3 and Rab Orchestrating Regulated Secretion - Pp. Rab5, Rab21, and Rab22 - Pp. Rab11a, Rab8a and Myosin V: Regulators of Recycling and Beyond - Pp. Roland and Lynne A. Lapierre View Abstract Purchase Chapter. Transport from Late Endosomes to the Golgi: Rab9 GTPase - Pp.