Rab GTPases and Membrane Trafficking

Ypt/Rab GTPases form the broadest group of GTPases in eukaryotic cells. These conserved GTPases are key regulators of intracellular trafficking. Like other monomeric GTPases, Ypt/Rabs switch between GDP- and GTP-bound forms with the help of their upstream regulators. When in the GTP-bound form, they interact with downstream effectors that mediate all vesicular transport steps, from vesicle formation to fusion. In addition, Ypt/Rabs are considered as candidates to coordinate intermediate/individual steps during vesicular transport. Due to their central role in intracellular traffic regulation, Ypt/Rabs have been implicated in human diseases ranging from diabetes and cancer to neurological and immunological disorders.

The Golgi apparatus is the main sorting compartment of the secretory pathway. At the cis Golgi cisterna, proteins are transported from the endoplasmic reticulum (ER) and sorted for forward transport or for recycling back to the ER. At the trans Golgi cisterna, proteins arriving from the medial Golgi cisterna or from endosomes are sorted for transport to the plasma membrane (PM) or to endosomes. In yeast, the Ypt1 and functional pair Ypt31/32 GTPases are required for transport at the cis and trans Golgi, respectively. The mammalian homologues of Ypt1 and Ypt31/32, Rab1 and Rab11, respectively, play similar roles. Therefore, these GTPases are considered the Golgi gatekeepers. Here, we summarize our current knowledge of upstream regulation and downstream effectors of the Golgi Ypt/Rab GTPases. In addition, we discuss the role of the Golgi Ypt/Rab GTPases in human health and disease.

Rab6 belongs to the most conserved Rab GTPases. It localizes to the Golgi apparatus and cytoplasmic vesicles and interacts with numerous effectors, including golgins and vesicle tethering complexes, adaptor proteins, as well as microtubule-based and actin-based motors. Rab6 regulates trafficking from endosomes to the Golgi apparatus, from the Golgi to ER and to the plasma membrane, as well as intra-Golgi transport and Golgi homeostasis. Recent studies demonstrate that Rab6 can coordinate all the sequential events underlying transport between two compartments, including budding/fission, transport and docking/tethering of membrane carriers.

Sec4p is one of the founding members of the Rab family of small GTPases, which controls post- Golgi exocytosis in the budding yeast Saccharomyces cerevisiae. Studying Sec4p in the facile yeast genetic system has provided important insight into the molecular mechanisms of Rab proteins function and regulation. Sec4p is activated by its guanine nucleotide exchange factor Sec2p, which is in turn recruited by Ypt31p and Ypt32p, the upstream Rab proteins that regulate vesicle budding from the tans-Golgi network. The downstream effectors of Sec4p include the exocyst, which mediates vesicle tethering, and the lgl proteins Sro7/77p that regulate SNARE assembly and membrane fusion. In addition, activation of Sec4p is required for the engagement of secretory vesicles to actin cables for directional transport of cargos to the daughter cell. Thus, through the control of exocytosis machinery and interaction with actin cytoskeleton, Sec4p plays an important role in asymmetric cell growth in budding yeast.

A subset of Rab GTPases have instrumental roles in the biogenesis, trafficking, docking and exocytosis of secretory granules, secretory lysosomes and synaptic vesicles. The four Rab3 isoforms and the two Rab27 isoforms are the main members of this family of “secretory Rabs.” Redundancy between the isoforms and between the Rab3 and Rab27 proteins has made the functional characterization of these proteins difficult. Data collected from different cell types suggest that the main role of Rab27 is to promote the recruitment of secretory vesicles at the release sites, while that of Rab3 is to control the number of ready-to-fuse vesicles. However, many observations that cannot be incorporated into this simplified scheme suggest that Rab3 and Rab27 have overlapping functions at different stages of the “life cycle” of secretory vesicles. Consistent with this, while some effector molecules are specific for Rab3 or Rab27, several interact with both of them.

Melanocytes and cytotoxic T lymphocytes are the two cell types where the functional importance of the Rab GTPases Rab27a was first convincingly shown. Indeed, Rab27a is a resident Rab in the signature organelles present in these two cell types - the melanosome in the melanocyte and the lytic granule in the T cell. For both cell types, loss of Rab27a results in a striking defect in the function of their signature organelle. In the case of the melanocyte, Rab27a in the melanosome membrane serves to connect the organelle to the actin cytoskeleton by binding to its effector protein melanophilin, which in turn binds to the actin-based motor protein myosin Va. This myosin-based interaction of the melanosome with the actin cytoskeleton in the periphery of the melanocyte serves, together with long-range, bidirectional, microtubuledependent movement of the organelle, to drive the proper intracellular distribution of melanosomes required for normal pigmentation in mammals. Delineation of this mechanism was aided enormously by the positional cloning of the mouse coat color genes encoding Rab27a (ashen), melanophilin (leaden) and myosin Va (dilute). Interestingly, a related tripartite complex containing Rab27a, its effector protein MyRip/Slac2-c, and myosin VIIa connects melanosomes to the actin cytoskeleton in retinal pigmented epithelial cells. In the case of the T cell, Rab27a in the membrane of the lytic granule is required for the docking and fusion of the granule at the immunological synapse, which in turn is required for target cell killing. Intense efforts are underway to define the downstream effectors of Rab27a in T cells that mediate this essential function. These efforts have been aided to a very large extent by the identification of several genes mutated in a family of closely-related lymphocyte diseases referred to as Familial Hemophagocytic Lymphohistiocytosis. Those studies have identified Munc13-4, a member of a family of proteins involved in controlling SNARE pairing, as one key downstream effector of Rab27a in T cells. Other studies have suggested that the Rab27a effector proteins Slp1 and Slp2a may facilitate the docking of lytic granules via their C2 domains.

Early steps of endocytosis involve budding and formation of endocytic vesicles from the plasma membrane, movement of the vesicles along cytoskeleton, and fusion with early endosomes. The Rab5 subfamily of GTPases (Rab5, Rab21, and Rab22) are localized to early endosomes and plasma membrane and play an important role in regulation of early endocytosis and signal transduction, via interactions with multiple effector proteins. Rab5, Rab21, and Rab22 share some common effectors, but differentially interact with other effectors and regulators, and exhibit distinct functions in early endocytosis. The interplay of the early endocytic Rabs is illustrated by the Rab22-Rab5 cascade in which Rab22-GTP recruits Rabex-5 (a Rab5 GEF) to early endosomes for activation of Rab5 and stimulation of early endosome fusion. This and other functions of Rab5, Rab21, and Rab22 in the formation and uncoating of endocytic vesicles, the vesicle movement on cytoskeleton, and the early endosome fusion are discussed in this review.

The endosomal system is a mosaic of highly dynamic interconnected organelles that serve many physiological functions including nutrient uptake, regulation of cell polarity, migration and plasma membrane remodeling. These widely divergent processes rely on the flawless transfer of cargo molecules between the distinct endosomal subcompartments and is regulated by members of the rab family of small GTPases and their effector networks. Here we provide an overview of the function of rab4, and the machinery it deploys to regulating exocytic membrane trafficking from early endosomes.

Recycling of endocytic cargoes utilizes multiple pathways employing Rab small GTPases. Rab11a is associated with slow recycling pathways that are responsible for plasma membrane recycling in non-polarized cells and the apical recycling system in polarized cells. Rab11a assembles multiple protein complexes along the recycling pathway utilizing multiple Rab11-Family Interacting Proteins and Class V myosins. Rab8a also assembles regulatory complexes associated with recycling pathways. While myosin V species are involved in both Rab11a- and Rab8a-dependent pathways, the details of interactions between these trafficking pathways remains unclear.

Late endosomal biogenesis depends on the Rab7 GTPase and its interaction with effectors. Within this review, we will focus on the Rab7 activation and inactivation cycle, and identify the critical regulators and interaction partners. We will highlight the role of Rab7 in membrane tethering between late endosome and lysosome, and its function in nutrient sensing, which is coupled to the establishment of membrane contact zones and late endosomal distribution within the cell.

Rab9 functions in the retrieval of mannose 6-phosphate receptors (MPRs) from late endosomes and their subsequent delivery to the trans Golgi network (TGN). In this chapter, we will discuss how Rab9 is recruited onto membranes, how Rab9 functions to select and segregate MPRs into a specific microdomain depleted of Rab7, and ultimately, how Rab9-containing transport vesicles dock and fuse at the TGN.

Rab GTPases have been identified more than 20 years ago, and their central role as regulators of protein trafficking has attracted considerable attention to this protein family. Hundreds of Rabs have been described in the literature based on bioinformatics analysis, but only a small proportion has been experimentally characterized. Using the human Rab family as an example, we discuss here how our knowledge of the Rab universe is biased towards evolutionarily older, more highly and widely expressed proteins. Newly described Rab proteins have thus received little attention. We explore the types of functional and structural novelty that newly characterized Rabs are unveiling, and discuss the importance of these poorly characterized proteins by exploring their participation in human disease.