A coiled-coil domain of melanophilin is essential for Myosin Va recruitment and melanosome transport in melanocytes

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F-binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.     

Darkness descends with two Rabs

Over 60 distinct Rab GTPases regulate specific vesicular transport steps in the mammalian central vacuolar system. Wasmeier et al. (this issue, p. 271) reveal a redundant role for two tissue-specific Rab proteins in regulating transport to a tissue-specific lysosome-related organelle, the melanosome.     

Rab27 Effectors,Pleiotropic Regulators in Secretory Pathways

Rab27, a member of the small GTPase Rab family, is widely conserved in metazoan, and two Rab27 isoforms, Rab27A and Rab27B, are present in vertebrates. Rab27A was the first Rab protein whose dysfunction was found to cause a human hereditary disease, type 2 Griscelli syndrome, which is characterized by silvery hair and immunodeficiency. The discovery in the 21st century of three distinct types of mammalian Rab27A effectors [synaptotagmin‐like protein (Slp), Slp homologue lacking C2 domains (Slac2), and Munc13‐4] that specifically bind active Rab27A has greatly accelerated our understanding not only of the molecular mechanisms of Rab27A‐mediated membrane traffic (e.g. melanosome transport and regulated secretion) but of the symptoms of Griscelli syndrome patients at the molecular level. Because Rab27B is widely expressed in various tissues together with Rab27A and has been found to have the ability to bind all of the Rab27A effectors that have been tested, Rab27A and Rab27B were initially thought to function redundantly by sharing common Rab27 effectors. However, recent evidence has indicated that by interacting with different Rab27 effectors Rab27A and Rab27B play different roles in special types of secretion (e.g. exosome secretion and mast cell secretion) even within the same cell type. In this review article, I describe the current state of our understanding of the functions of Rab27 effectors in secretory pathways .     

The Rab GTPase family

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

Sec15 is an effector for the Rab11 GTPase in mammalian cells

Rab/Ypt GTPases play key roles in the regulation of vesicular trafficking. They perform most of their functions in a GTP-bound form by interacting with specific downstream effectors. The exocyst is a complex of eight polypeptides involved in constitutive secretion and functions as an effector for multiple Ras-related small GTPases, including the Rab protein Sec4p in yeast. In this study, we have examined the localization and function of the Sec15 exocyst subunit in mammalian cells. Overexpressed Sec15 associated with clusters of tubular/vesicular elements that were concentrated in the perinuclear region. The tubular/vesicular clusters were dispersed throughout the cytoplasm upon treatment with the microtubule-depolymerizing agent nocodazole and were accessible to endocytosed transferrin, but not exocytic cargo (vesicular stomatitis virus glycoprotein). Consistent with these observations, Sec15 colocalized selectively with the recycling endosome marker Rab11 and exhibited a GTP-dependent interaction with the Rab11 GTPase, but not with Rab4, Rab6, or Rab7. These findings provide the first evidence that the exocyst functions as a Rab effector complex in mammalian cells.     

Rab-dependent cellular trafficking and amyotrophic lateral sclerosis

Rab GTPases are becoming increasingly implicated in neurodegenerative disorders, although their role in amyotrophic lateral sclerosis (ALS) has been somewhat overlooked. However, dysfunction of intracellular transport is gaining increasing attention as a pathogenic mechanism in ALS. Many previous studies have focused axonal trafficking, and the extreme length of axons in motor neurons may contribute to their unique susceptibility in this disorder. In contrast, the role of transport defects within the cell body has been relatively neglected. Similarly, whilst Rab GTPases control all intracellular membrane trafficking events, their role in ALS is poorly understood. Emerging evidence now highlights this family of proteins in ALS, particularly the discovery that C9orf72 functions in intra transport in conjunction with several Rab GTPases. Here, we summarize recent updates on cellular transport defects in ALS, with a focus on Rab GTPases and how their dysfunction may specifically target neurons and contribute to pathophysiology. We discuss the molecular mechanisms associated with dysfunction of Rab proteins in ALS. Finally, we also discuss dysfunction in other modes of transport recently implicated in ALS, including nucleocytoplasmic transport and the ER-mitochondrial contact regions (MAM compartment), and speculate whether these may also involve Rab GTPases.     

Rab GTPases and myosin motors in organelle motility

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

The role of ARF and Rab GTPases in membrane transport.

Two key events of intracellular transport and membrane trafficking in eukaryotic cells, the formation of transport vesicles and their specific delivery to target membranes, are controlled by small GTPases of the ADP-ribosylation factor (ARF) and Rab families, respectively. The past 18 months have seen the identification of proteins that regulate ARF and Rab GDP/GTP cycle, as well as the characterization of their effectors, shedding light on the molecular mechanisms of ARF and Rab function.     

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome‐related organelle that is commonly defective in Hermansky–Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC‐1, ‐2, ‐3, or AP‐1, ‐3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A‐depletion phenotype resembles Rab38/32‐inactivated or BLOC‐3‐deficient melanocytes, suggesting that Rab9A works in line with BLOC‐3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC‐3‐deficiency in melanocytes decreased the length of STX13‐positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co‐regulatory GTPases control STX13‐mediated cargo delivery to maturing melanosomes.     

Use of the two-hybrid system to identify Rab-interacting proteins

The yeast two-hybrid system has been useful for identifying many partners and effectors of small GTPases of the Rab family. We describe here such a screen using Rab6, a protein involved in the regulation of intracellular transport at the level of the Golgi apparatus, as bait.     

Griscelli syndrome: a model system to study vesicular trafficking

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutations in either the myosin VA (GS1), RAB27A (GS2) or melanophilin (GS3) genes. The three GS subtypes are commonly characterized by pigment dilution of the skin and hair, due to defects involving melanosome transport in melanocytes. Here, we review how detailed studies concerning GS have contributed to a better understanding of the molecular mechanisms involved in vesicle transport and membrane trafficking processes. Additionally, we demonstrate that the identification and biological analysis of novel disease‐causing mutations highlighted the functional importance of the RAB27A‐MLPH‐MYO5A tripartite complex in intracellular melanosome transport. As the small GTPase Rab27a is able to interact with multiple effectors, including Slp2‐a and Myrip, we report on their presumed role in melanosome transport. Furthermore, we summarize data suggesting that RAB27B and RAB27A are functionally redundant and hereby provide further insight into the pathogenesis of GS2. Finally, we discuss how the gathered knowledge about the RAB27A‐MLPH‐MYO5A tripartite complex can be translated into a possible therapeutic application to reduce (hyper)pigmentation of the skin.     

Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases

Rab27A is required for actin-based melanosome transport in mammalian skin melanocytes through its interaction with a specific effector, Slac2-a/melanophilin. Mutations that disrupt the Rab27A/Slac2-a interaction cause human Griscelli syndrome. The other Rab27 isoform, Rab27B, also binds all of the known effectors of Rab27A. In this study, we determined the crystal structure of the constitutively active form of Rab27B complexed with GTP and the effector domain of Slac2-a. The Rab27B/Slac2-a complex exhibits several intermolecular hydrogen bonds that were not observed in the previously reported Rab3A/rabphilin complex. A Rab27A mutation that disrupts one of the specific hydrogen bonds with Slac2-a resulted in the dramatic reduction of Slac2-a binding activity. Furthermore, we generated a Rab3A mutant that acquires Slac2-a binding ability by transplanting four Rab27-specific residues into Rab3A. These findings provide the structural basis for the exclusive association of Slac2-a with the Rab27 subfamily, whereas rabphilin binds several subfamilies, including Rab3 and Rab27.     

Evidence for defective Rab GTPase-dependent cargo traffic in immune disorders

A fully functional immune system is essential to protect the body against pathogens and other diseases, including cancer. Vesicular trafficking provides the correct localization of proteins within all cell types, but this process is most exquisitely controlled and coordinated in immune cells because of their specialized organelles and their requirement to respond to selected stimuli. More than 60 Rab GTPases play important roles in protein trafficking, but only five Rab-encoding genes have been associated with inherited human disorders, and only one of these (Rab27a) causes an immune defect. Mutations in RAB27A cause Griscelli Syndrome type 2 (GS2), an autosomal recessive disorder of pigmentation and severe immune deficiency. In lymphocytes, Munc13-4 is an effector of Rab27a, and mutations in the gene encoding this protein (UNC13D) cause Familial Hemophagocytic Lymphohistiocytosis Type 3 (FHL3). The immunological features of GS2 and FHL3 include neutropenia, thrombocytopenia, and immunodeficiency due to impaired function of cytotoxic lymphocytes. The small number of disorders caused by mutations in genes encoding Rabs could be due to their essential functions, where defects in these genes could be lethal. However, with the increasing use of next generation sequencing technologies, more mutations in genes encoding Rabs may be identified in the near future.     

Purification and identification of novel Rab effectors using affinity chromatography

Rab GTPases are central regulatory elements of the intracellular transport machinery of eukaryotic cells. To regulate vesicle docking and fusion as well as organelle dynamics Rab proteins interact with effector molecules in the GTP-bound active state. The identification of Rab effectors is, therefore, of primary importance for the mechanistic understanding of intracellular transport. Here we describe the experimental system we have developed to biochemically purify and identify effectors of the small GTPase Rab5. The method, which is based on an affinity chromatography procedure, results in the large-scale purification of Rab effectors in amounts sufficient for both their identification by microsequencing techniques and their functional characterization. In the case of Rab5, the procedure allows a comprehensive analysis of the downstream effectors and regulators of this GTPase. We expect this strategy to provide fundamental insights into the molecular mechanism of membrane transport but also to be applicable to several other GTPase-dependent biological functions.     

Rab7 and Rab27a control two motor protein activities involved in melanosomal transport

Melanosomes are lysosome-related organelles that synthesize, store and transport melanin. In epidermal melanocytes, melanosomes mature and are transferred to surrounding keratinocytes, which is essential for skin and coat colour. Mouse coat colour mutants reveal a critical role for the small GTPase Rab27a, which recruits myosin Va through its effector protein melanophilin/Slac2a. Here we have studied how two different Rab GTPases control two motor proteins during subsequent phases in transport of melanosomes. We show that the small GTPase Rab7 mainly associates with early and intermediate stage melanosomes and Rab27a to intermediate and mature melanosomes. Rab27a is found in an active state on mature melanosomes in the tips of the dendrites. The Rab7-Rab7-interacting lysosomal protein-dynein pathway only controls early and intermediate stage melanosomes because the mature melanosomes lack Rab7 and associate with the actin network through Rab27a recruited MyoVa. Thus two Rab proteins regulate two different motor proteins, thereby controlling complementary phases in melanosome biogenesis: Rab7 controls microtubule-mediated transport of early and Rab27a the subsequent actin-dependent transport of mature melanosomes.     

Distinct Rab27A binding affinities of Slp2-a and Slac2-a/melanophilin: Hierarchy of Rab27A effectors

The small GTPase Rab27A has recently been shown to regulate melanosome transport in mammalian skin melanocytes through sequentially interacting with two Rab27A effectors, Slac2-a/melanophilin and Slp2-a. Although Slac2-a and Slp2-a contain a similar N-terminal Rab27A-binding domain (named SHD, Slp homology domain), nothing is known about the functional differences between the Slac2-a SHD and Slp2-a SHD. In this study, the Rab27A-binding affinity of ten putative Rab27A effector proteins has been investigated. It has been found that they could be classified into a low-affinity group (e.g., Slac2-a) and a high-affinity group (e.g., Slp2-a and Slp4-a) based on their Rab27A-binding affinity. Kinetic analysis of the GTP-Rab27A-binding to the SHD of Slp2-a, Slp4-a, and Slac2-a by surface plasmon resonance further indicated that the kinetic parameters of Rab27A for the Slp2-a SHD, Slp4-a SHD, and Slac2-a SHD consisted of a fast association rate constant (3.35 x 10(4), 2.06 x 10(4), and 2.11 x 10(4) M(-1) s(-1), respectively) and a slow dissociation rate constant (4.48 x 10(-4), 3.96 x 10(-4), and 2.37 x 10(-3) s(-1) respectively), and their equilibrium dissociation constants were determined to be 13.4, 19.2, and 112 nM, respectively. Our data suggest that distinct Rab27A binding activities of Slac2-a and Slp2-a ensure the order (or hierarchy) of Rab27A effectors that sequentially function in melanosome transport in melanocytes.     

Semi-automated analysis of organelle movement and membrane content: understanding rab-motor complex transport function

Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the movement of organelles and associated fluorescent proteins, and automatic organelle tracking, to compare organelle movement parameters with the association of membrane proteins. This approach allowed large-scale, unbiased analysis of the contribution of organelle-associated proteins and cytoskeleton tracks in motility. Using this strategy, we addressed the role of membrane recruitment of Rab GTPases and effectors in organelle dynamics, using the melanosome as a model. We found that Rab27a and Rab32/38 were mainly recruited to sub-populations of slow-moving/static and fast-moving melanosomes, respectively. The correlation of Rab27a recruitment with slow movement/docking was dependent on the effector melanophilin. Meanwhile, using cytoskeleton-disrupting drugs, we observed that this speed:Rab content relationship corresponded to a decreased frequency of microtubule (MT)-based transport and an increased frequency of actin-dependent slow movement/docking. Overall, our data indicate the ability of Rab27a and effector recruitment to switch melanosomes from MT- to actin-based tethering and suggest that a network of Rab signalling may integrate melanosome biogenesis and transport.     

Extensive GTPase crosstalk regulates Golgi trafficking and maturation

Virtually all transport events at the Golgi complex are regulated by Arf and Rab family GTPases. Recent work has advanced our knowledge regarding the mechanisms controlling GTPase activity, and it has become clear that GTPases do not act in isolation but rather function in complex networks of crosstalk and feedback. Together with earlier findings, these recent studies indicate that communication between GTPases, their regulatory proteins, effectors, and lipids plays a pivotal role in Golgi transport and cisternal maturation.     

Characterization of the molecular defects in Rab27a,caused by RAB27A missense mutations found in patients with Griscelli syndrome

Rab27a plays a pivotal role in the transport of melanosomes to dendrite tips of melanocytes and mutations in RAB27A, which impair melanosome transport cause the pigmentary dilution and the immune deficiency found in several patients with Griscelli syndrome (GS). Interestingly, three GS patients present single homozygous missense mutations in RAB27A, leading to W73G, L130P, and A152P transitions that affect highly conserved residues among Rab proteins. However, the functional consequences of these mutations have not been studied. In the present report, we evaluated the effect of overexpression of these mutants on melanosome, melanophilin, and myosin-Va localization in B16 melanoma cells. Then we studied several key parameters for Rab27a function, including GTP binding and interaction with melanophilin/myosin-Va complex, which links melanosomes to the actin network. Our results showed that Rab27a-L130P cannot bind GTP, does not interact with melanophilin, and consequently cannot allow melanosome transport on the actin filaments. Interestingly, Rab27a-W73G binds GTP but does not interact with melanophilin. Thus, Rab27a-W73G cannot support the actin-dependent melanosome transport. Finally, Rab27a-A152P binds both GTP and melanophilin. However, Rab27a-A152P does not allow melanosome transport and acts as a dominant negative mutant, because its overexpression, in B16 melanoma cells, mimics a GS phenotype. Hence, the interaction of Rab27a with melanophilin/myosin-Va is not sufficient to ensure a correct melanosome transport. Our results pointed to an unexpected complexity of Rab27a function and open the way to the search for new Rab27a effectors or regulators that control the transport of Rab27a-dependent vesicles.