Rab molecular switches are key players in defining membrane identity and regulating intracellular trafficking events in eukaryotic cells. In spite of their global structural similarity, Rab-family ...members acquired particular features that allow them to perform specific cellular functions. The overall fold and local sequence conservations enable them to utilize a common machinery for prenylation and recycling; while individual Rab structural differences determine interactions with specific partners such as GEFs, GAPs and effector proteins. These interactions orchestrate the spatiotemporal regulation of Rab localization and their turning ON and OFF, leading to tightly controlled Rab-specific functionalities such as membrane composition modifications, recruitment of molecular motors for intracellular trafficking, or recruitment of scaffold proteins that mediate interactions with downstream partners, as well as actin cytoskeleton regulation. In this review we summarize structural information on Rab GTPases and their complexes with protein partners in the context of partner binding specificity and functional outcomes of their interactions in the cell.
Rab GTPases control intracellular membrane traffic by recruiting specific effector proteins to restricted membranes in a GTP-dependent manner. In this Cell Science at a Glance and the accompanying ...poster, we highlight the regulation of Rab GTPases by proteins that control their membrane association and activation state, and provide an overview of the cellular processes that are regulated by Rab GTPases and their effectors, including protein sorting, vesicle motility and vesicle tethering. We also discuss the physiological importance of Rab GTPases and provide examples of diseases caused by their dysfunctions.
Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, ...however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.
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•Ribonucleoprotein particles are associated with endosomes in axons•Rab7a endosomes provide sites for axonal local translation•Rab7a endosomes support axonal synthesis of survival factors•CMT2B-Rab7a mutations affect axonal translation and mitochondrial integrity
Late endosomes are sites of local translation of mRNAs important for mitochondrial maintenance, and perturbing this process can contribute to neuropathology, as seen in Charcot Marie-Tooth disease.
Rab35 is one of the first discovered members of the large Rab GTPase family, yet it received little attention for 10 years being considered merely as a Rab1‐like GTPase. In 2006, Rab35 was recognized ...as a unique Rab GTPase localized both at the plasma membrane and on endosomes, playing essential roles in endocytic recycling and cytokinesis. Since then, Rab35 has become one of the most studied Rabs involved in a growing number of cellular functions, including endosomal trafficking, exosome release, phagocytosis, cell migration, immunological synapse formation and neurite outgrowth. Recently, Rab35 has been acknowledged as an oncogenic GTPase with activating mutations being found in cancer patients. In this review, we provide a comprehensive summary of known Rab35‐dependent cellular functions and detail the few Rab35 effectors characterized so far. We also review how the Rab35 GTP/GDP cycle is regulated, and emphasize a newly discovered mechanism that controls its tight activation on newborn endosomes. We propose that the involvement of Rab35 in such diverse and apparently unrelated cellular functions can be explained by the central role of this GTPase in regulating phosphoinositides and F‐actin, both on endosomes and at the plasma membrane.
In the past 10 years, Rab35 has become one of the most studied Rabs involved in a growing number of key cellular functions, including endosomal trafficking, exosome release, phagocytosis, cell migration, immunological synapse formation and neurite outgrowth. In addition, oncogenic activating mutations have been recently found in cancer patients. Here, we provide a comprehensive review on how Rab35 controls these apparently unrelated functions by regulating phosphoinositides and F‐actin, both on endosomes and at the plasma membrane.
Of the approximately 70 human Rab GTPases, nearly three-quarters are involved in endocytic trafficking. Significant plasticity in endosomal membrane transport pathways is closely coupled to receptor ...signaling and Rab GTPase-regulated scaffolds. Here we review current literature pertaining to endocytic Rab GTPase localizations, functions, and coordination with regulatory proteins and effectors. The roles of Rab GTPases in (1) compartmentalization of the endocytic pathway into early, recycling, late, and lysosomal routes; (2) coordination of individual transport steps from vesicle budding to fusion; (3) effector interactomes; and (4) integration of GTPase and signaling cascades are discussed.
Melanosomes are specialized intracellular organelles that produce and store melanin pigments in melanocytes, which are present in several mammalian tissues and organs, including the skin, hair, and ...eyes. Melanosomes form and mature stepwise (stages I–IV) in melanocytes and then are transported toward the plasma membrane along the cytoskeleton. They are subsequently transferred to neighboring keratinocytes by a largely unknown mechanism, and incorporated melanosomes are transported to the perinuclear region of the keratinocytes where they form melanin caps. Melanocytes also extend several dendrites that facilitate the efficient transfer of the melanosomes to the keratinocytes. Since the melanosome biogenesis, transport, and transfer steps require multiple membrane trafficking processes, Rab GTPases that are conserved key regulators of membrane traffic in all eukaryotes are crucial for skin and hair pigmentation. Dysfunctions of two Rab isoforms, Rab27A and Rab38, are known to cause a hypopigmentation phenotype in human type 2 Griscelli syndrome patients and in chocolate mice (related to Hermansky–Pudlak syndrome), respectively. In this review article, I review the literature on the functions of each Rab isoform and its upstream and downstream regulators in mammalian melanocytes and keratinocytes.
The life of melanosomes from biogenesis in a melanocyte to degradation in a keratinocyte. Abbreviations: MS, melanosome; MTs, microtubules; AFs, actin filaments. The insets show the Rab32/38–Varp complex required for step (1) and the Rab27A–Mlph/Slac2‐a–myosin Va transport complex required for step (3).
Mutations in leucine‐rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). Recent studies have shown that LRRK2 physiologically phosphorylates several Rab family proteins including ...Rab12 and that this phosphorylation is accelerated by the pathogenic mutations in LRRK2, although the significance in the PD pathogenesis remains unknown. Here we examined the effect of the overexpression of LRRK2 on the distribution of organelles in cultured cells and found that lysosomes become clustered in a perinuclear region upon the overexpression of pathogenic mutant LRRK2 in a manner dependent on its kinase activity. The perinuclear clustering of lysosomes was abolished by knocking out RAB12 as well as its effector protein RILPL1. Re‐expression of Rab12 in RAB12 knockout cells suggested that the phosphorylation at Ser106 of Rab12 is required for the perinuclear clustering of lysosomes. Moreover, phosphorylated Rab12 was also accumulated on the clustered lysosomes, and the phosphorylation of Rab12 increased its interaction with RILPL1, leading us to conclude that the increase in the phosphorylation of Rab12 by pathogenic LRRK2 compromised intracellular lysosomal transport via the enhanced interaction of Rab12 with RILPL1. These data suggest the involvement of abnormal regulation of lysosomal transport in the LRRK2‐mediated pathogenesis of PD.
Mutations in LRRK2 cause familial Parkinson's disease. LRRK2 phosphorylates Rab12, which is accelerated by pathogenic mutations. We found that lysosomes clustered in a perinuclear region upon overexpression of the pathogenic mutant LRRK2. This clustering was abolished by knocking out Rab12 and its effector protein RILPL1. Phosphorylation of Rab12 at Ser106 facilitated its interaction with RILPL1 and was required for the induction of perinuclear lysosomal clustering. This suggests that abnormal regulation of lysosomal transport is involved in LRRK2‐mediated Parkinson's disease pathogenesis.
Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which ...links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.
Rab family small GTPases are master regulators of distinct steps of intracellular vesicle trafficking in eukaryotic cells. GDP‐bound cytoplasmic forms of Rab proteins are prone to aggregation due to ...the exposure of hydrophobic groups but the machinery that determines the fate of Rab species in the cytosol has not been elucidated in detail. In this study, we find that BAG6 (BAT3/Scythe) predominantly recognizes a cryptic portion of GDP‐associated Rab8a, while its major GTP‐bound active form is not recognized. The hydrophobic residues of the Switch I region of Rab8a are essential for its interaction with BAG6 and the degradation of GDP‐Rab8a via the ubiquitin‐proteasome system. BAG6 prevents the excess accumulation of inactive Rab8a, whose accumulation impairs intracellular membrane trafficking. BAG6 binds not only Rab8a but also a functionally distinct set of Rab family proteins, and is also required for the correct distribution of Golgi and endosomal markers. From these observations, we suggest that Rab proteins represent a novel set of substrates for BAG6, and the BAG6‐mediated pathway is associated with the regulation of membrane vesicle trafficking events in mammalian cells.
Synopsis
BAG6 specifically recognizes exposed hydrophobic residues in the Switch I region of inactive, GDP‐bound Rab8a. BAG6 targets GDP‐Rab8a to proteasomal degradation to maintain the integrity of the Golgi and endosomal compartments.
BAG6 is required for the correct distribution of Golgi/endosomal markers.
BAG6 recognizes the GDP‐bound form of Rab8a for ubiquitin‐mediated degradation.
The hydrophobicity of the Switch I region is essential for the instability of Rab8a.
BAG6 specifically recognizes exposed hydrophobic residues in the Switch I region of inactive, GDP‐bound Rab8a. BAG6 targets GDP‐Rab8a to proteasomal degradation to maintain the integrity of the Golgi and endosomal compartments.
We previously reported that Parkinson's disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). ...Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.
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