Rab32 subfamily small GTPases: pleiotropic Rabs in endosomal trafficking

Abstract

Rab small GTPases, well-known regulators of membrane trafficking pathways in eukaryotic cells, comprise approximately 60 different members in mammals. During the past decade, our understanding of the functions of mammalian Rab32 subfamily members (Rab32 and Rab38) have deepened, especially on the biogenesis of lysosome-related organelles, such as melanosomes, and the protection mechanisms against several pathogenic microbial infections. Endosome-mediated membrane trafficking by Rab32 subfamily members plays pivotal roles in these events. In this review, we provide an overview of the regulatory mechanisms of mammalian Rab32-family members in endosomal trafficking, especially focusing on their GEF, GAP and effector molecules, and describe the latest findings on physiological and pathological functions regulated by these molecules.

GAP, GEF, lysosome-related organelles, membrane traffic, Rab

Rab small GTPases belong to the Ras superfamily and are constituted of approximately 60 family members in mammals. Each member of Rab family localizes at distinct organelles/subcellular compartments and play pivotal roles in intracellular membrane trafficking events, including cargo sorting, vesicle budding, vesicle formation, vesicle transport, and the docking, tethering and fusion of vesicles with target membranes in eukaryotic cells (1). Like other small GTPases, cycling between a GTP-bound active and a GDP-bound inactive form of Rab proteins is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively (2, 3). Upon extracellular stimulation of cells, Rab proteins are activated by GEFs, which enable binding with their effector molecules to promote membrane trafficking events (4). Recently, a considerable attention has been directed to the endosomal functions of two closely related Rabs, Rab32 and Rab38 (hereafter called Rab32-subfamily members) in mammals, because these Rabs or their regulators have been shown to be involved not only in biogenesis of lysosome-related organelles (LROs) but also in defense against certain microbial infection. In this review, we first briefly describe what is ‘endocytic networks’ and what is ‘LROs’ and then give an overview of functions and regulation of mammalian Rab32-subfamily members especially in the endosomal trafficking as shown in Fig. 1.

Fig. 1

Schematic diagram of endosomal trafficking systems regulated by Rab32-family members. The cycling of Rab32/Rab38 between the active and inactive forms plays pivotal roles in the biogenesis of the melanosome. In the early endosome (left lower figure), AP-3 regulates the sorting and transport of Tyr, and BLOC-1/BLOC-2 regulate the transport of Tyrp1. BLOC-1 has been shown to be an effector of Rab5, a well-known Rab existing on the early endosome (52). BLOC-2 functions as an effector of Rab32/Rab38 and promotes the stabilization of Stx13-positive intermediate tubular carriers (right lower figure) (53). VAMP7 forms a complex with t-SNARE (i.e. Stx3 and SNAP23) locating on the melanosome, thereby playing an essential role in the transport of melanosomal cargoes (e.g. Tyr/Tyrp1) (right lower figure) (54). In parallel, there is a model in which VAMP7 on the melanosome forms a complex with Stx13 on the tubular carrier containing the melanosomal cargoes to transport the melanosomal cargoes (right lower figure) (53). Since the function of VAMP7 is suppressed by the Rab32/Rab38-Varp complex (26), Rab32/Rab38 need to be inactivated presumably by RUTBC1 before the tubular carrier containing melanosomal cargoes fuses to the melanosome (right lower figure). VAMP7 carried on the melanosome along with Tyr/Tyrp1 is recycled back to the early endosome by the Rab32/Rab38-Varp complex, which is newly activated by BLOC-3 (right upper figure). Both BLOC-3 and RUTBC1 are thought to be recruited to the melanosome via Rab9 existing on the early endosomes/melanosomes (31, 35). Thus, the Rab32/Rab38 cycling regulated by BLOC-3 and RUTBC1 is likely to play a central role in the transport of melanosome cargoes.

Internalized proteins, such as receptors and cell adhesion molecules, taken up from the plasma membrane are transported to the early endosome, where the proteins are sorted out and destined to the degradation or recycling pathway. The trafficking of epidermal growth factor receptor (EGFR) is a typical example of the degradation pathway: activated EGFR is transported to the lysosome via the late endosome to be degraded. On the other hand, the trafficking of recycling cargoes, such as transferrin receptor and integrin, is regulated by the recycling pathway, in which the recycling cargoes are transported by the recycling endosome, a tubular membrane carrier emanating from the early endosome and responsible for the cargo recycling back to the plasma membrane (5). In addition, the retromer-mediated trafficking pathway has been shown to mediate the endosome to the trans-Golgi network (TGN) (called retrograde transport) or to the plasma membrane (6). In this way, complicated transport systems starting from the early endosome are constructed in eukaryotic cells. Interestingly, the trafficking pathway from the early/recycling endosomes to LROs also exists (see below for details).

Melanosome, a Representative LROs

LROs are a series of cell type-specific intracellular membrane compartments. Although LROs share some features with endosomes and lysosomes, such as acidic pH and certain luminal and membrane components, they also have an unique morphology and composition to exert their own functions, including pigmentation, pulmonary homeostasis, and immunity in mammals (7). The melanosome is a well-known representative LRO, where melanin pigments are produced and stored, in melanocytes (in mammals) and melanophores (in other vertebrates, such as fish and amphibians). It contains a series of lysosomal proteins including lysosome-associated membrane protein (LAMP)1–LAMP3 and its lumen pH is acidic, like other LROs, e.g. the dense granule in platelets and the lytic granule in cytotoxic T lymphocytes and natural killer cells (7). Besides these lysosomal proteins, biogenesis of the melanosome requires melanosome-specific proteins/enzymes, which are transported from the early/recycling endosomes by membrane trafficking systems. A number of relevant factors for the melanosome biogenesis have been identified by a series of genetic analyses of human diluted pigment disorders and their corresponding model animals. Abnormal melanosome biogenesis and transport are known to cause a number of diseases with diluted hair, skin, and eye colour, such as Chédiak–Higashi syndrome, Hermansky–Pudlak syndrome (HPS), and Griscelli syndrome (8). Among them, in the melanocytes from HPS patients and their corresponding model animals, the melanosome biogenesis is impaired, and several causative genes for HPS have been reported. Based on their clinical features, the proteins encoded by at least nine types of HPSs (HPS1–9) reported are classified into three groups: adaptor protein complex AP-3 (impaired in HPS2), biogenesis of lysosome-related organelles complex-1 (BLOC-1, impaired in HPS7–9), BLOC-2 (impaired in HPS3, 5 and 6) and BLOC-3 (impaired in HPS1, and 4) (9, 10). Interestingly, each group of proteins functions as a protein complex, and the dysfunction of a single subunit of the complexes leads to the disruption of functional integrity of each protein complex. Because the trafficking pathway of the melanogenic enzymes, including tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1), from the early/recycling endosomes to melanosomes is inhibited in BLOCs-deficient melanocytes, BLOC complexes have been thought to be post early endosome regulators. However, the subunits composing BLOC complexes do not have any conserved protein motifs, which so far prevents understanding their precise functions in the melanosome biogenesis. Recently, using very fine live-cell imaging, the functions of these BLOC complexes have been gradually revealed. For example, BLOC-1 and BLOC-2 have been shown to facilitate exit of melanosomal cargoes, especially melanogenic enzymes such as Tyrp1, from the early/recycling endosomes into tubular carriers and from tubular carriers to the melanosome, respectively (11, 12). However, because BLOCs are known to be expressed ubiquitously, the exact reason why the dysfunctions of BLOC complexes cause tissue/organ-specific disorders such as diluted pigmentation remains to be elucidated.

Functions of Rab32/Rab38 in the Melanosome Biogenesis

Rab32/Rab38 in various animal species

During the past decade, a number of researches points to the critical roles of two closely related Rab small GTPases, Rab32 and Rab38, in the regulation of LROs. These two Rabs with similar functions have approximately 75% identity in their sequences and are known to be specifically expressed in LRO-containing tissues/cells, including the melanocyte and platelet, albeit that Rab32 exhibits a broader expression profile than Rab38 (13). A spontaneous mouse mutant with HPS-like phenotypes, chocolate, shows weakly diluted coat colour and impaired secretion of lung surfactant proteins, and the responsible gene for chocolate has been identified as Rab38 (14). Such a mild dilution phenotype of chocolate mice can be explained by the compensation effect of Rab32, because Rab32/Rab38 double-deficient melanocytes exhibit more severe hypopigmentation by blocking the trafficking of melanogenic enzymes to the melanosome (15). In other organisms, Rab38 gene is mutated in Ruby rats with the HPS-like phenotypes, such as hypopigmentation and platelet defects (16). In the melanophores of fish and amphibians, melanosome movement along the microtubule is precisely regulated by extracellular stimuli, such as Melatonin and α-melanocyte-stimulating hormone. In Xenopus laevis, Rab32 is localized on the melanosome and plays a pivotal role in the stimuli-dependent melanosome movement (17). In Danio rerio, due to teleost-specific genome duplication, there are two Rab32 (Rab32a and Rab32b) and three Rab38 (Rab38a, Rab38b and Rab38c) isoforms. Rab32a is expressed in the notochordal cells where it regulates formation of notochord vacuoles that are also known as LROs. Because the notochord vacuoles are required for body axis morphogenesis, zebrafish expressing an inactivated form of Rab32a exhibits abnormal somitic pattern and vertebrae (18). Rab38a mRNA is expressed in the pigmented cells, which suggests that Rab38 is also involved in the pigmentation in zebrafish (19). In invertebrate, the function of Rab32/Rab38 homolog has been reported in a simple chordate, ascidian Ciona intestinalis (20). Fibroblast growth factor (FGF) signal plays an essential role in the differentiation of anterior sensory pigment cells in ascidian embryos. The expression level of Rab32/Rab38 ortholog increases upon FGF signal activation and the Rab32/Rab38 ortholog plays an essential role in sensory pigment cell formation in ascidian embryos. GLO-1 is a Rab32/Rab38 ortholog in C.elegans; it is required for the biogenesis of gut granules that are considered LROs (21). Furthermore, Rab-related protein 1, a Drosophila melanogaster ortholog of Rab32/Rab38, is expressed on eye pigment granules that are also considered LROs, and its mutation causes eye hypopigmentation (22). Taken together, Rab32 and Rab38 are considered to play pivotal roles in the biogenesis of LROs in various animal species ranging from invertebrates to vertebrates.

Rab32/Rab38 effectors

VPS9-ankyrin-repeat protein (Varp), originally identified as a Rab21-GEF, is a Rab32/Rab38 effector molecule and was shown to be expressed in a wide variety of tissues (23). As its name indicates, Varp has an N-terminal vacuolar protein sorting 9 (VPS9) domain and two ankyrin repeat domains (ANKR1 and ANKR2) in the C-terminal region. The ANKR1 domain is responsible for the activated Rab32/Rab38 binding, thereby enabling formation of Varp–Rab32/Rab38 complex, which promotes the trafficking of melanosome cargoes, such as Tyr/Tyrp1/dopachrome tautomerase (Dct), to the melanosome in melanocytes (24, 25, 35). Moreover, Varp binds to vesicle-associated membrane protein 7 (VAMP7, also known as tetanus neurotoxin-insensitive VAMP, TI-VAMP) via a region between the ANKR1 and the ANKR2 domains (VAMP7-interaction domain, VID) (23). Owen and colleagues have demonstrated that the interaction between Varp and VAMP7 prevents the SNARE complex formation with t-SNARE molecules (26). More recently Marks and colleagues have proposed an exciting model regarding the new mode of SNARE recycling (11): they found that activated Rab32/Rab38 recruits Varp on the melanosome and that the Rab32/Rab38–Varp complex inhibits the function of VAMP7, which enables the reuse of VAMP7 for the next round of the melanosomal cargo trafficking (Fig. 1). Taken together, these reports indicate that the Varp–Rab32/Rab38 complex has at least two functions: the promotion of trafficking of Tyr/Tyrp1/Dct from the early/recycling endosomes to the melanosome and the recycling of VAMP7 from the melanosome to the early/recycling endosomes.

Varp is not a sole Rab32/Rab38 effector molecule in melanocytes, and De Pietro and colleagues have reported that myosin Vc is another Rab32/Rab38 effector molecule (27). Class V actin-based motor proteins are known to regulate vesicle/organelle transport along the actin cytoskeleton, and three different isoforms of myosin V (myosin Va, Vb and Vc) are present in mammals. Myosin Va is known to promote trafficking of the mature melanosome along the cortical actin filaments through indirect binding to activated Rab27A on the mature melanosome. The binding of myosin Va–Rab27A is mediated by a linker protein, Slac2-a/melanophilin (8), and loss of any one of the components of the myosin-Va–Slac2-a–Rab27A complex results in the hypopigmentation of Griscelli syndrome (28). Myosin Vb functions with Rab8 and Rab11 to regulate several types of recycling pathways (29). Myosin Vc localizes mainly on the early/recycling endosomes, where activated Rab32/Rab38 exist. Interaction between myosin Vc and Rab32/Rab38 has been proposed to regulate trafficking of melanosomal cargoes such as Tyrp1 and VAMP7 to the melanosome (27). Moreover, it has been shown that the BLOC-2 is a Rab32/Rab38 effector and participates in targeting recycling tubular carriers containing the melanogenic enzymes to the melanosome (Fig. 1) (12). These reports collectively indicate that tissue-specific Rab32 and Rab38 regulate multiple steps of the trafficking of melanosomal cargoes together with ubiquitously expressing their effectors from endosomes to the melanosome, and vice versa.

Regulation of Rab32/Rab38

In contrast to a variety of Rab32/Rab38-effectors, a small number of their regulators have been reported to date. BLOC-3 is a protein complex composed of HPS1 and HPS4, both of which are most frequently mutated in HPS patients. Barr and colleagues have demonstrated that BLOC-3 functions as a Rab32/Rab38-GEF, and the melanosomal cargo Tyrp1 is not correctly transported to melanosomes in HPS1- and/or HPS4-deficient melanocytes, thereby inhibiting the melanosome maturation and pigmentation (30). Moreover, HPS4 has been shown to be an effector molecule of Rab9 (31). In general, Rab9 is known to be predominantly localized at the late endosome and to regulate the retrograde transport of cargo proteins such as mannose-6-phosphate receptors from the late endosome to the TGN in non-melanocytic cells (32). More recently, however, Rab9 has also been shown to exist in the early/recycling endosomes and the melanosome and has been suggested to be involved in the activation of Rab32/Rab38 on Rab9-localizing membranes by recruiting BLOC -3 (33).

Tre-2/Bub2/Cdc16 (TBC)-domain-containing molecules are generally thought to be GAPs for Rab small GTPases (2). RPIP8/UNC-14/NESCA (RUN) and TBC domain-containing protein 1 (RUTBC1), originally identified as a Rab9 effector molecule, has been shown to possess GAP activity against Rab32 and Rab33B in an in vitro GAP assay (34). Recently, we found that RUTBC1 functions as a Rab32/Rab38-GAP in melanocytes (35): in RUTBC1-overexpressing melanocytes, Rab32/Rab38 are inactivated, and thereby the melanosomal cargoes (tyrosinase/Tyrp1/Dct) fails to be transported to the melanosome. In line with the model that interactions between Rabs and their effector molecules are thought to be important for determining intracellular localization of Rabs (36), we observed that the complex composed of Rab9 and RUTBC1 localizes on the melanosome, although Rab9 or RUTBC1 expressed alone is observed mainly in the late endosome/lysosome or in the cytosol. Furthermore, we found that the trafficking of melanosomal cargoes (tyrosinase/Tyrp1/Dct) is inhibited in the RUTBC1-knockdown melanocytes even though Rab32/Rab38 are hyper-activated and accumulated on the melanosome membrane. These findings suggest that the proper spatiotemporal regulation of Rab32/Rab38 is required for the normal trafficking of melanosomal cargoes.

Functions of Rab32/Rab38 in Regulating Intracellularly Invaded Pathogens

It is well known that pathogenic microorganisms invade mammalian epithelial cells through phagocytosis, a special type of endocytosis in mammalian cells (37). Salmonella enterica is a member of the Enterobacteriaceae family and known to be a cause of foodborne illness. S. enterica can invade a wide variety of mammalian species and produce intracellular vacuoles called SCV (Salmonella-containing vesicle), where several types of Rab small GTPases (i.e., Rab5, Rab7 and Rab9) are known to be recruited (38). Because the SopB gene product encoded by Salmonella exhibits a structural similarity to phosphatidylinositol 4-phosphatase and has an ability to produce phosphatidylinositol-3-phosphate (PI3P) on the SCV membrane (39), the SCV has a phospholipid component similar to that of the early endosome. It is generally thought that substances in the early endosome, e.g. internalized bacteria, are degraded by being transported to the lysosome via the late endosome, where Rab7 and Rab9 play important roles in promoting lysosomal maturation and in trafficking of the lysosomal proteases from the TGN to the late endosome/lysosome, respectively (40). To avoid such Rab7/Rab9-mediated maturation of the SCV, Salmonella secrete specific effector proteins. Actually, it has been reported that a Salmonella effector SifA inhibits the functions of Rab7 and Rab9 for internalized Salmonella to escape from the host defense systems (40, 41).

It should be noted that S. enterica comprises numerous variants, and some of them show different host specificites. For example, Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A) cause typhoid fever only in humans but not in rodents such as mice, whereas S. Typhimurium can infect a wide range of hosts including humans and mice. The different host specificities appear to be related to the Salmonella effector-dependent degradation of Rab32, which localizes on the SCV membrane (42). Intriguingly, the GtgE gene product encoded by S. Typhimurium exhibits proteolytic activity toward Rab32. The resulting reduction in the amount of the Rab32 protein on the SCV enables S. Typhimurim to replicate in many hosts (42). Moreover, when S. Typhimurim GtgE was ectopically expressed in S. Typhi, which specifically infects humans, the pathogen acquired the ability to replicate in mouse-derived macrophages (42). The same group has recently reported that the SopD2 gene product, another effector of S. Typhimurim, is also able to inhibit the recruitment of host Rab32 to the SCV without affecting the total amount of Rab32 (43). Because SopD2 possesses GAP activity against Rab32 as well as Rab8/Rab10, it probably inhibits activation of Rab32 and its recruitment onto the SCV. Thus, S. Typhimurim is likely to break the host defense mechanism using the two effectors, GtgE and SopD. In line with this evidence, S. Typhi can be replicated in the BLOC-3 mutant mice, in which Rab32/Rab38 are not activated (42).

Listeria monocytogenes is an intracellular parasitic bacterium and known as a causative pathogen of listeriosis with meningoencephalitis, sepsis and abortion (44). L. monocytogenes can be internalized into host cells such as dendritic cells by phagocytosis and then induces endosome-like vacuoles called LCV (listeria-containing vesicle). After that, L. monocytogenes is able to invade into the host cytoplasm by piercing the LCV membrane with liseriolysin O, an effector of the pathogen, to escape from phagolysosomal degradation (44). Recently, Rab32 and its effectors, PHB/PHB-2, have been shown to be involved in the degradation of intracellular L. monocytogenes by enclosing the pathogen escaping from the LCV into the cytoplasm (45). This process seems to be somewhat similar to that of macroautophagy, although the Rab32-mediated enclosing process of L. monocytogenes does not involve the Atg5-mediated autophagy pathway (44). Because, in another literature, Rab32 has been reported to be involved in the autophagy process (46), future research is needed to unravel the detailed relationship between the canonical autohphagy and the Rab32-mediated containment of L. monocytogenes.

As with other bacteria, the genus Mycobacterium is known to be taken into host macrophages by phagocytosis and encompasses a number of species that cause several serious diseases. Especially, M. leprae and M. tuberculosis are well known to cause leprosy and tuberculosis, respectively. Leprosy is a chronic granulomatous infection affecting both skin and peripheral nerves. A large-scale genome-wide association study concerning leprosy has shown that Rab32 is one of the susceptible genes (47). Another study on Mycobacterium has shown that several Rab proteins including Rab32 and Rab38 localize on the M. tuberculosis-containing phagosome (48). In the literature, Rab32 and Rab38 are considered to regulate the trafficking of cathepsin D, a lysosomal protease, to the M. tuberculosis-containing phagosome; however, the precise roles of Rab32/Rab38 in the regulation of M. lepare and M. tuberculosis are still obscure.

Hepatitis C virus (HCV) is an important risk factor for chronic liver diseases, such as liver cirrhosis and hepatocellular carcinoma (49). It has very recently been reported that Rab32 is involved in the hepatocellular infection of HCV. In transcriptome analysis using hepatocellular carcinoma cells Huh7.5 infected with HCV, the Rab32 mRNA level was found to be elevated. In addition, HCV infection in Huh7.5 cells has been shown to promote the conversion of Rab32 from a GTP-bound active form to a GDP-bound inactive form by an unknown mechanism. Interestingly, the core protein of HCV has a potential to bind with GDP-Rab32 in the endoplsmic reticulum (ER), which makes it possible that HCV assembles around the ER to escape from host defense using the endolysosomal system (50).

Concluding Remarks and Perspectives

In this article, we reviewed current knowledge on the functions of mammalian Rab32 subfamily members, their effectors, and their regulators in the endosomal trafficking, especially focusing on the biogenesis of LROs and the host defense against pathogen infection. In the biogenesis of LROs, tissue-specific Rab32 and Rab38 regulate the trafficking of LRO cargoes from the early/recycling endosomes to LROs through binding ubiquitously expressing effector molecules/complexes, including Varp, myosin-Vc and BLOC-2. In addition, the new mode of SNARE recycling from LROs has been identified (11). Although the precise mechanism(s) of host defense regulated by Rab32 is still not clear as compared to the case of LROs, since Rab32 subfamily members promote the trafficking of Tyr/Tyrp1/Dct via the endosomal trafficking pathway as mentioned above, it is highly possible that some antibacterial substances are transported to the bacteria-containing vesicle in a Rab32/Rab38-dependent manner. It is of great interest that production of melanin in insects is known to play a central role to defend against pathogens (51). Future understanding of the Rab32-mediated host defense systems should become more important both in academic and medical fields. Furthermore, future research would deepen the integrated understanding of the commonality and uniqueness of Rab32 subfamily members' functions in the LRO biogenesis and host defense.

Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Grant-in-Aid for Scientific Research (C) JP15K07039 to N. O., Grant-in-Aid for Scientific Research (B) JP15H04367 and Grant-in-Aid for Scientific Research on Innovative Areas JP16H01189 to M. F., and Grant-in-Aid for Scientific Research (A) JP15H02503 to Y. K.) and by the Cosmetology Research Foundation (to N.O.).

Conflict of Interest

None declared.

Abbreviations

Abbreviations
 
• ANKR

  ankyrin repeat

 
• AP

  adaptor protein complex

 
• BLOC

  biogenesis of lysosome-related organelles complex

 
• Dct

  dopachrome tautomerase

 
• ER

  endoplasmic reticulum

 
• GAP

  GTPase-activating protein

 
• GEF

  guanine nucleotide exchange factor

 
• HCV

  hepatitis C virus

 
• HPS

  Hermansky–Pudlak syndrome

 
• LAMP

  lysosome-associated membrane protein

 
• LCV

  listeria-containing vesicle

 
• RILP

  Rab7-interacting lysosomal protein

 
• RUN

  RPIP8/UNC-14/NESCA

 
• RUTBC1

  RUN and TBC1

 
• SCV

  Salmonella-containing vesicle

 
• TBC

  Tre-2/Bub2/Cdc16

 
• TGN

  trans-Golgi network

 
• Tyr

  tyrosinase

 
• Tyrp1

  tyrosinase-related protein 1

 
• VAMP

  vesicle-associated membrane protein

 
• Varp

  VPS9-ankyrin-repeat protein

 
• VID

  VAMP7-interaction domain

 
• VPS9

  vacuolar protein sorting 9