Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

RAB Family

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101912

Synonyms

Historical Background and Introduction

RAB proteins are evolutionarily highly conserved monomeric small GTP-binding proteins, which form the largest branch within the RAS superfamily. The first RAB gene Sec4/Ypt (yeast protein transcript) was identified more than 30 years ago and later described as the gene of a small G protein having essential function in vesicle trafficking between the Golgi apparatus and the plasma membrane (Gallwitz et al. 1983). Only a few years later, the first mammalian family members were cloned and named as RAB (Ras-like in rat brain) proteins (Touchot et al. 1987). In humans, more than 70 different RAB and RAB-like proteins have been characterized so far, and the protein family has also numerous members in all the well-known model organisms (11 in Saccharomyces cerevisiae, 29 in Caenorhabditis elegans, 33 in Drosophila melanogaster, and 57 in Arabidopsis thaliana). All RAB family proteins share common structural features and – through their downstream effectors – orchestrate vesicle trafficking, docking, and fusion events in eukaryotic cells.

Protein Structure and Upstream Regulation

Despite their small size (21–25 kDa), RAB proteins comprise several highly conserved regions and interaction domains. All of the RABs contain a nucleotide-binding domain characteristic for GTPases and able to bind both GTP and GDP. Although the amino acid sequence of different RAB proteins shows 55–75% identity, there are divergent regions predominantly at the amino and carboxy terminals. The hypervariable carboxy-terminal region is required for the correct subcellular localization of the protein (Stenmark et al. 1994). This region contains two cysteines usually prenylated by RAB geranylgeranyl transferases, thus facilitating their attachment to cell membranes. However, since the variable carboxy-terminal region of RABs does not contain a consensus sequence, geranylgeranyl transferases are not able to specifically recognize them. Instead, RAB escort protein (REP) binds to a more conserved region of RABs and presents the proteins to RAB geranylgeranyl transferase (Gomes et al. 2003).

In their tertiary structure, RABs possess a common GTPase fold: 5 α-helixes surrounding the central barrel formed by a six-stranded β-sheet. They generally cycle between GTP-bound active and GDP-bound inactive forms, where binding of the given nucleotide determines the structure of their switch I and II regions. In GDP-bound state, switch regions are rather disordered, whereas GTP binding results in the rearrangement of switch regions into a well-ordered state. These structural changes explain the altered affinity of RAB proteins toward their specific effectors and upstream regulators upon GDP or GTP binding (Pfeffer 2005).

The general cycling of RABs between GTP-bound active and GDP-bound inactive forms is controlled by upstream regulators (Fig. 1). As RABs have a low intrinsic hydrolase activity, their hydrolysis rate depends on GTPase activating proteins (GAPs). These proteins can complement the catalytic site of RABs and promote GTP hydrolysis. Thus, GAPs act as negative regulators of RAB proteins. Inactive, GDP-bound RABs are removed from their target membrane and kept soluble in the cytoplasm by GDP dissociation inhibitors (GDIs). As a first step for RAB activation, GDIs are removed, mainly by membrane-associated GDI displacement factors (GDFs). Thereafter, guanine nucleotide exchange factors (GEFs) activate RAB proteins by stimulating the exchange of GDP to GTP. In their active state, RABs can stably bind to membrane surfaces via their C-terminal geranylgeranyl anchor and recruit various effectors to the membrane they are attached to (Stenmark 2009).
RAB Family, Fig. 1

Cycling of RAB proteins between GTP-bound active and GDP-bound inactive state is controlled by upstream regulators. GTPase activating proteins (GAPs) facilitate GTP hydrolysis, thereby contributing to RAB inactivation. GDP-bound RABs are removed from their target membrane and sequestrated in the cytosol by GDP dissociation inhibitors (GDIs). As a first step of RAB activation, GDIs are removed by GDI displacement factors (GDFs), and then guanine nucleotide exchange factors (GEFs) stimulate the exchange of GDP to GTP. Active, GTP-bound RABs recruit various effectors to the membrane they are attached to via a geranylgeranyl anchor

Subcellular Localization and Function

RABs are peripheral membrane proteins localized to the cytosolic face of specific intracellular membranes, where they function as master regulators of several membrane trafficking events. Different RAB proteins were found to be responsible for orchestrating the single steps of vesicle budding, actin- and microtubule-dependent vesicle movement, docking, and membrane fusion. As RABs accumulate on the membrane of their target compartment in steady state, they specify membrane identity; this is why they are widely used as markers for different organelles (Table 1). Although most RABs are ubiquitously expressed, some show a cell-type or tissue-specific distribution. For instance, RAB3A is expressed only by neurons, where it is attached to synaptic vesicles. RAB13 and RAB17 show epithelial expression, whereas RAB27A is a protein product of hematopoietic cell lines (Bhuin and Roy 2014).
RAB Family, Table 1

Subcellular localization and function of the best studied human RAB proteins

Protein

Localization

Function

RAB1

ER-Golgi intermediate compartment

ER-Golgi anterograde transport

RAB2

ER-Golgi intermediate compartment

Golgi-ER retrograde transport

RAB3

Secretory vesicles

Exocytosis, neurotransmitter release

RAB4

EEs

Direct recycling to plasma membrane (PM)

RAB5

EEs

Endocytosis, early endosomal fusion, early steps of endosome maturation

RAB6

Golgi

Intra-Golgi transport, transport to the PM, Golgi-ER retrograde transport

RAB7

LEs, autophagosomes/amphisomes

Endosome maturation, motility and lysosomal fusion

RAB8

TGN, GLUT4 vesicles

Transport to the PM

RAB9

LEs

LE-TGN transport

RAB10

GLUT4 vesicles

Transport to the PM

RAB11

REs, post-Golgi vesicles

Transport to the PM, indirect endocytic recycling

RAB13

Tight junctions, TGN

Transport to the PM

RAB14

EEs, GLUT4 vesicles

Transport to the PM

RAB15

EEs and REs

Endocytic recycling

RAB18

Lipid droplets

Lipid droplet formation

RAB21

EEs

Integrin internalization

RAB22

EEs, TGN

Bidirectional EE-TGN transport

RAB23

EEs, PM

Recycling to the PM

RAB24

ER, cis-Golgi

Autophagosome formation

RAB25

Apical REs

Apical RE-PM transport

RAB26

TGN, apico-lateral membranes

Exocytosis of secretory granules and vesicles

RAB27A

Melanosomes, secretory granules

Transport toward the PM, exocytosis

RAB32

Melanosomes, mitochondria

Mitochondrial fission, autophagy, melanosome biogenesis

RAB33

Golgi

Intra-Golgi trafficking, autophagy

RAB34

Macropinosomes

Macropinocytosis

RAB35

REs

Apical endocytic recycling

RAB37

Secretory granules

Exocytosis

RAB38

Melanosomes

TGN-melanosome transport, melanosome biogenesis

As key regulators of vesicle trafficking, different RAB proteins are attached to vesicles of the secretory pathway. RAB1 mediates the vesicular transport of newly synthetized proteins from the endoplasmic reticulum (ER) to cis-Golgi, whereas RAB2 plays a part in retrograde Golgi-ER transport. These proteins can be found predominantly on the vesicles of the pre-Golgi intermediate compartment. Intra-Golgi trafficking is mediated by the Golgi-localized RAB6, RAB33, and RAB40. RAB8 plays an important role in constitutive biosynthetic vesicle trafficking from trans-Golgi network (TGN) to the plasma membrane (PM), in glucose transporter type 4 (GLUT4) vesicle translocation (together with RAB10 and RAB14), and in ciliogenesis (with RAB23). Various types of exocytosis are mediated by RAB3, RAB26, RAB27, and RAB37.

RABs also have well-defined roles in the endocytic pathway. RAB5 is often used as a marker of early endosomes (EEs), although it is localized to phagosomes and caveosomes as well. It is involved in the homotypic fusion of primer endocytic vesicles into EEs and also regulates macropinocytosis (with RAB34) and maturation of early phagosomes (together with RAB14 and RAB22). The endocytic recycling toward the PM is mediated by RAB4 on a direct route and by RAB11 and RAB35 through recycling endosomes (REs). RAB7 is localized to late endosomes (LEs) and is required for endosome maturation, transport, and lysosomal fusion. Endocytic and secretory routes have numerous connections. For instance, RAB22 ensures bidirectional vesicle transport between EEs and TGN, whereas trafficking from LEs to TGN is mediated by the primarily late endosomal RAB9 (Stenmark 2009).

As autophagic membrane can be derived from various intracellular membrane sources depending on cell type, RAB proteins play a significant role in membrane delivery from different organelles to the pre-autophagosomal structure (PAS). RAB24 and the Golgi-localized RAB33 were shown to participate in autophagosome formation. RAB1 and RAB32 play also a part in the initial steps of autophagy, presumably by providing membrane source from the ER and ER-mitochondria contact sites (RAB32 is involved in the process of mitochondrial fission as well). Moreover, RAB11 was recently found to be involved in both autophagosome formation and maturation events (Szatmári and Sass 2014).

Melanosomes are lysosome-related organelles, which are responsible for the synthesis, storage, and transport of melanin. RAB32 and RAB38 play a key role in melanosome biogenesis, whereas RAB27 is also required for the translocation of melanosomes toward the cell periphery. RAB18, as a regulator of lipid droplet formation, is often used as a marker to follow the dynamics of lipid metabolism. RAB13 controls the assembly of tight junctions between epithelial cells (Bhuin and Roy 2014).

Role in Vesicle Trafficking

Active, membrane-attached RAB proteins recruit various effectors to their target membranes, which facilitate different steps of vesicle trafficking process (Hutagalung and Novick 2011) (Fig. 2). RAB proteins can orchestrate cargo selection and vesicle budding. Mannose-6-phosphate receptors (MPRs) deliver newly synthetized lysosomal enzymes from the TGN to degradative compartments, such as LEs, but instead being degraded, they are recycled and transported back to the TGN in a RAB9-dependent manner. On LEs, RAB9 interacts with its effector, TIP47, which specifically binds the cytoplasmic domain of MPRs and collects them on the site of the forming transport vesicle.
RAB Family, Fig. 2

RAB proteins orchestrate the different steps of membrane trafficking. Through their effector proteins, they can mediate vesicle budding and cargo recruitment into the vesicles. Some RABs interact with phosphatidylinositol (PI) kinases and phosphatases, so they change the PI content and the identity of the membranes, facilitating uncoating of vesicles. RAB proteins can interact with motor proteins directly or indirectly, thus promoting vesicle movement along the cytoskeleton. By recruiting tethering factors and SNARE proteins, RABs actively control vesicle docking and membrane fusion

Other RABs facilitate vesicle movement along actin filaments or microtubules. A well-studied example is RAB11, which – through its effector, RAB11-FIP2 – recruits myosin Vb motor protein to REs, thus mediating their transport toward the PM. Similarly, in yeast, Ypt11p controls the transport of mitochondria and Golgi from mother cell to the bud by interacting with a type V myosin. RABs also participate in microtubule-based vesicle transport toward both the periphery and the centrosome, depending on its binding partners. The late endosomal RAB7 interacts with Rab-interacting lysosomal protein (RILP), which subsequently recruits the dynein-dynactin motor complex. As a consequence, LEs are transported toward the centrosome, where they undergo fusion with lysosomes. On the other hand, the Golgi-localized RAB6 binds directly Rabkinesin-6, which moves toward the cell periphery, facilitating this way anterograde intra-Golgi trafficking.

Before vesicles could fuse with their target membranes, coat proteins must be removed from them. Some RABs have important role in this process. For instance, RAB5 has a key role in uncoating primer endocytic vesicles. It recruits PI 4,5 phosphatases and phosphatidylinositol 3 kinases (PI3K), thus stimulating the turnover of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), a lipid marker of PM and essential for the binding of AP-2 clathrin adaptor complex. By enhancing the lipid content change of the vesicular membrane, RAB5 promotes the disassembly of clathrin coat and recycling of the coat proteins.

After uncoating, vesicles are transported into the close proximity of their target membranes and attached to it by mono- or multimeric tethering factors. These tethering proteins are also RAB effectors, which keep the membranes close to each other and control the subsequent steps of fusion. One of the best studied examples is the multisubunit homotypic fusion and vacuolar protein sorting (HOPS) complex. During endosome maturation, RAB7 recruits the HOPS subunits to the endosomal membrane, which also act as a RAB7 GEF; thereby, they generate a positive feedback loop stabilizing and further enhancing RAB7 activity. In parallel, HOPS complex performs its tethering function and interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which eventually mediate membrane fusion. Furthermore, a HOPS subunit directly regulates the fusion event by facilitating SNARE complex assembly.

RABs not only specify the identity of organelle membranes, but – by acting in cascades – they have a crucial role in changing membrane identity. Later, it was thought that the different organelles of secretory and endocytic routes are spatially separated and only vesicular transport ensures the connection between them. However, a growing number of studies show that these organelles undergo a maturation process during which their soluble content and membrane proteins and lipids are changing; thus, they are dynamically transforming into each other. An outstanding example is the process of endosome maturation, which is orchestrated by the switch between RAB5 and RAB7 proteins. On EEs, RAB5 is accumulated and through its effectors – such as the PI3K Vps34 which generates the high phosphatidylinositol 3-phosphate (PI3P) content of EE membrane – it creates the specific membrane identity of these organelles. RAB5 also enhances the activity of its own GEF, generating a positive feedback loop, which provides enough time for protein sorting and recycling, primary role of EEs. Among RAB5 effectors there is class C core vacuole/endosome tethering (CORVET) complex, which is different from the RAB7 effector HOPS only in two subunits. However, RAB5 also recruits Mon1/Ccz1 proteins, which initiate RAB conversion by replacing RAB5 with RAB7 and CORVET-specific subunits with the HOPS-specific ones (especially those with RAB7 GEF activity). Thus, the RAB content of the endosomal membrane is gradually changing, and the growing amount of RAB7, together with its specific effectors, ensures transition of EEs into LEs (Poteryaev et al. 2010).

Clinical Significance

Owing to the great physiological importance of RAB proteins, not only in membrane trafficking but – by controlling receptor turnover – also in the regulation of signaling pathways, their mutations and malfunction are associated with various human diseases, such as neurological and endocrinological diseases, cancer, or infections (Corbeel and Freson 2008).

Several RABs were found to have an important role in neurodegenerative diseases, which are characterized by abnormal accumulation of protein aggregates, causing neuronal loss and other severe symptoms. In models of Parkinson’s disease, RAB1 overexpression reduced the disruptive effect of mutant, aggregate-prone α-synuclein on ER-Golgi trafficking (Cooper et al. 2006; Winslow et al. 2010). In Huntington’s disease, mutant huntingtin protein was shown to prevent RAB8 interaction with its effectors, thus disrupting post-Golgi traffic targeted for lysosomes (del Toro et al. 2009). Huntingtin also binds a RAB11 GEF, leading to the accumulation of inactive RAB11 and impaired recycling toward the PM (Li et al. 2009). Disruption of exocytotic neurotransmitter release or endocytosis in neurons leads to different forms of neuropathy. A rare form of X-linked mental retardation is caused by mutations of GDI1, probably responsible for impairment in RAB3 function required for exocytosis of synaptic vesicles. Moreover, RAB7 mutations are associated with another type of neuropathy, Charcot-Marie-Tooth disease type 2 (Corbeel and Freson 2008).

A form of partial albinism called Griscelli syndrome is associated with dysfunction of RAB27 or its effectors, causing disruption of actin-based movement of melanosomes. RAB27A is also affected in choroideremia, an X chromosome-linked form of progressive blindness characterized by the degeneration of retinal pigment epithelium. In choroideremia patients, mutations of REP1 result in insufficient prenylation and dysfunction of RAB27A.

If glucose transporter type 4 (GLUT4) is translocated to the PM, it mediates insulin-dependent glucose uptake of adipocytes and skeletal and heart muscle cells. RAB8A, RAB8B, RAB10, and RAB14 are found on GLUT4-containing vesicles, and, presumably, they have a role in their trafficking. In accordance, mutations of their GAPs AS160 (also known as TBC1D4) and TBC1D1 were described in type II diabetes patients (Stenmark 2009), and RAB10 itself was also proven to be involved in insulin-stimulated fusion of these vesicles with the PM.

By regulating the secretory and endo-lysosomal degradation pathways, RAB proteins strongly influence numerous signaling pathways. Hence, it is not surprising that aberrant RAB expression can contribute to tumorigenesis and tumor progression. RAB25 is often overexpressed in breast and ovarian cancers; and its ectopic expression increases proliferation and inhibits apoptotic cell death in cell culture. As RAB25 is involved in the endocytic recycling route, its overexpression probably increases the presence of signaling receptors in the PM. Furthermore, as an interaction partner of β1 integrin, RAB25 also contributes to the development of migration phenotype in tumor cells. RAB8 (which, among others, regulates matrix metalloprotease release), RAB21, and RAB23 were also found to be connected with tumor development (Goldenring 2013).

Many pathogens enter the host cell through the endocytic pathway, where they are able to exploit RABs and their effectors and hijack them to promote their own survival and replication. For example, group B coxsackievirus enters epithelial cells by triggering the internalization of tight junction proteins by RAB5 and RAB34. Similarly, Salmonella enterica subsp. enterica serovar Typhimurium exploits numerous RABs to facilitate its own uptake into phagosomes. Following phagosome maturation, engulfed pathogens are normally eliminated by lysosomal degradation. Thus bacteria, in order to avoid elimination, often hijack RAB proteins playing a part in endocytic maturation. For instance, mycobacteria, Listeria monocytogenes, and Salmonella typhimurium secrete factors to prevent RAB5 or RAB7 from being recruited to the bacterium-containing vacuole, thereby delaying phagosome maturation and avoiding lysosomal degradation. On the other hand, Legionella pneumophila targets RAB1 in infected alveolar macrophages by its own bacterial GEF, GDF, and GAP factors, creating a membrane-bound compartment with pre-Golgi intermediate compartment identity where bacteria can survive and replicate (Stenmark 2009).

Summary

RAB family proteins are small GTPases, which act as molecular switches cycling between GTP-bound active and GDP-bound inactive form. Their general cycle is dependent on upstream regulators. In their active state, they are attached to membranes of different organelles, where they recruit various effector proteins. Through their effectors, RABs specify membrane identity and regulate the main steps of vesicle budding, trafficking, and fusion events. As RABs are accumulated on their target membrane in steady state, they are often used as organelle biomarkers. Regarding their physiological significance, it is not surprising that mutations or dysfunctions of RAB proteins and their upstream regulators or effectors underlie several human disorders, such as neurological, metabolic, and infectious diseases or cancer.

See Also

References

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Anatomy, Cell and Developmental BiologyEotvos Lorand UniversityBudapestHungary