Rab-regulated interaction of early endosomes with lipid droplets

Recent studies indicate that lipid droplets isolated from a variety of different cells are rich in proteins known to regulate membrane traffic. Among these proteins are multiple Rab GTPases. Rabs are GTP switches that regulate intracellular membrane traffic through an ability to control membrane-membrane docking as well as vesicle motility. Here we present evidence that the multiple Rabs associated with droplets have a function in regulating membrane traffic. Droplet Rabs are removed by Rab GDP-dissociation inhibitor (RabGDI) in a GDP-dependent reaction, and are recruited to Rab-depleted droplets from cytosol in a GTP-dependent reaction. Rabs also control the recruitment of the early endosome (EE) marker EEA1 from cytosol. We use an in vitro reconstitution assay to show that transferrin receptor positive EEs bind to the droplet in a GTP/Rab-dependent reaction that appears not to lead to membrane fusion. This docking reaction is insensitive to ATP(gamma s) but is blocked by ATP. Finally, we show that when GTP bound active or GDP bound inactive Rab5 is targeted to the droplet, the active form recruits EEA1. We conclude that the Rabs associated with droplets may be capable of regulating the transient interaction of specific membrane systems, probably to transport lipids between membrane compartments.     

Rabs grab motors: defining the connections between Rab GTPases and motor proteins.

Rab GTPases and their effectors regulate membrane traffic by determining, along with cognate SNAREs, the specificity of transport vesicle docking and fusion steps. Recent studies have also implicated Rabs in the movement of these transport vesicles from their site of formation to their site of fusion, and several Rabs have been linked to specific microtubule- or actin-based motor proteins. Analyses of Rab and motor protein mutants, coupled with advanced imaging techniques, have led to the suggestion that certain Rabs function as essential components of the vesicle receptor for specific motor proteins.     

Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation

Lipid droplet is a cellular organelle with a neutral lipid core surrounded by a phospholipid monolayer and coated with structural as well as functional proteins. The determination of these proteins, especially their functional regulations and dynamic movement on and off droplets, holds a key to resolving the biological functions of the cellular organelle. To address this, we carried out a comprehensive proteomic study that includes a complete proteomic, a phosphoprotein proteomic, and a comparative proteomic analysis using purified lipid droplets and mass spectrometry techniques. The complete proteome identified 125 proteins of which 70 proteins had not been identified on droplets of mammalian cells previously. In phosphoprotein proteomic analysis, 7 functional lipid droplet proteins were determined to be phosphorylated, including adipose differentiation related protein (ADRP/ADFP), two Rab proteins, and four lipid metabolism enzymes, including adipose triglyceride lipase (ATGL). To understand the dynamics of lipid droplets, GTP-dependent protein recruitment was analyzed by comparative proteomics. Arf1 and some of its coatomers, three other Arfs, several other small G-proteins including 3 Rabs, and several lipid synthetic enzymes were recruited from cytosol to purified droplets. Together, the present study suggests that lipid droplet is an active and dynamic cellular organelle that governs lipid homeostasis and intracellular trafficking through protein phosphorylation as well as GTP-regulated protein translocation.     

Regulation of autophagy by the Rab GTPase network

Autophagy (macroautophagy) is a highly conserved intracellular and lysosome-dependent degradation process in which autophagic substrates are enclosed and degraded by a double-membrane vesicular structure in a continuous and dynamic vesicle transport process. The Rab protein is a small GTPase that belongs to the Ras-like GTPase superfamily and regulates the vesicle traffic process. Numerous Rab proteins have been shown to be involved in various stages of autophagy. Rab1, Rab5, Rab7, Rab9A, Rab11, Rab23, Rab32, and Rab33B participate in autophagosome formation, whereas Rab9 is required in non-canonical autophagy. Rab7, Rab8B, and Rab24 have a key role in autophagosome maturation. Rab8A and Rab25 are also involved in autophagy, but their role is unknown. Here, we summarize new findings regarding the involvement of Rabs in autophagy and provide insights regarding future research on the mechanisms of autophagy regulation.     

Large scale screening for novel rab effectors reveals unexpected broad Rab binding specificity

Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated. In this study we systematically screened for novel Rab effectors by a yeast two-hybrid assay with 28 different mouse or human Rabs (Rab1-30) as bait and identified 27 Rab-binding proteins, including 19 novel ones. We further investigated their Rab binding specificity by a yeast two-hybrid assay with a panel of 60 different GTP-locked mouse or human Rabs. Unexpectedly most (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms. As an example, inositol-polyphosphate 5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe) bound the greatest number of Rabs (i.e. 16 distinct Rabs). Others, however, specifically recognized only a single Rab isoform or only two closely related Rab isoforms. The interaction of eight of the novel Rab-binding proteins identified (e.g. INPP5E and Cog4) with a specific Rab isoform was confirmed by co-immunoprecipitation assay and/or colocalization analysis in mammalian cell cultures, and the novel Rab2B-binding domain of Golgi-associated Rab2B interactor (GARI) and GARI-like proteins was identified by deletion and homology search analyses. The findings suggest that most Rab effectors (or Rab-binding proteins) regulate intracellular membrane trafficking through interaction with several Rab isoforms rather than through a single Rab isoform.     

Small but mighty: Atg8s and Rabs in membrane dynamics during autophagy

Autophagy is a degradative pathway during which autophagosomes are formed that enwrap cytosolic material destined for turnover within the lytic compartment. Autophagosome biogenesis requires controlled lipid and membrane rearrangements to allow the formation of an autophagosomal seed and its subsequent elongation into a fully closed and fusion-competent double membrane vesicle. Different membrane remodeling events are required, which are orchestrated by the distinct autophagy machinery. An important player among these autophagy proteins is the small lipid-modifier Atg8. Atg8 proteins facilitate various aspects of autophagosome formation and serve as a binding platform for autophagy factors. Also Rab GTPases have been implicated in autophagosome biogenesis. As Atg8 proteins interact with several Rab GTPase regulators, they provide a possible link between autophagy progression and Rab GTPase activity. Here, we review central aspects in membrane dynamics during autophagosome biogenesis with a focus on Atg8 proteins and selected Rab GTPases.     

Ypt and Rab GTPases: insight into functions through novel interactions.

Ypt/Rab GTPases are key regulators of vesicular transport in eukaryotic cells. During the past two years, a number of new Ypt/Rab-interacting proteins have been identified and shown to serve as either upstream regulators or downstream effectors. Proteins that interact with these regulators and effectors of Ypt/Rabs have also been identified, and together they provide new insights into Ypt/Rab mechanisms of action. The picture that emerges from these studies suggests that Ypt/Rabs function in multiple and diverse aspects of vesicular transport. In addition, not only are Ypt/Rabs highly conserved, but their functions and interactions are as well. Interestingly, crosstalk among Ypt/Rabs and between Ypt/Rabs and other signaling factors, suggest the possibility of coordination of the individual vesicular transport steps and of the protein transport machinery with other cellular processes.     

A Ypt/Rab GTPase module makes a PAS

Organization of membrane micro-domains by Ypt/Rab GTPases is key for all membrane trafficking events in eukaryotic cells. Since autophagy is a membrane trafficking process, it was expected that these GTPases would play a role in autophagy as well. While evidence about participation of Ypt/Rabs in autophagy is beginning to emerge, the mechanisms by which they act in this process are still not clear. Moreover, it is still questionable if and how Ypt/Rabs coordinate autophagy with other cellular trafficking processes. Yeast Ypt1 and its mammalian homolog Rab1 are required for both endoplasmic reticulum (ER)-to-Golgi transport and autophagy, suggesting that they coordinate these two processes. In our recent paper, we identify Atg11, a bona fide phagophore assembly site (PAS) component, as a downstream effector of Ypt1. Moreover, we show that three components of a GTPase module—the Ypt1 activator, Trs85-containing TRAPP complex, Ypt1, and the Atg11 effector—interact on the PAS and are required for PAS formation during selective autophagy. We propose that Ypt/Rabs coordinate the secretory and the autophagic pathways by recruiting process-specific effectors.     

Insights into the role of the membranes in Rab GTPase regulation

Rab GTPases are molecular switches with essential roles in mediating vesicular trafficking and establishing organelle identity. The conversion from the inactive, cytosolic to the membrane-bound, active species and back is tightly controlled by regulatory proteins. Recently, the roles of membrane properties and lipid composition of different target organelles in determining the activity state of Rabs have come to light. The investigation of several Rab guanine nucleotide exchange factors (GEFs) has revealed principles of how the recruitment via lipid interactions and the spatial confinement on the membrane surface contribute to spatiotemporal specificity in the Rab GTPase network. This paints an intricate picture of the control mechanisms in Rab activation and highlights the importance of the membrane lipid code in the organization of the endomembrane system.     

Rab proteins: The key regulators of intracellular vesicle transport

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes.     

General role of GDP dissociation inhibitor 2 in membrane release of Rab proteins: modulations of its functional interactions by in vitro and in vivo structural modifications.

The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins in the functional cycle and recycling of Rab GTPases. Previous studies have demonstrated that GDI-1 can operate with multiple Rab proteins. In this study we have addressed a plausible general activity of GDI-2 in supporting Rab membrane release and have analyzed the requirements of sequence-conserved vs variable regions of GDI-2 in these functional interactions. The in vitro function of expressed recombinant GDI-2 wild-type-, point-, or deletion-mutant proteins was investigated toward several Rab family members, divergent in structure, localized and operating on different membranes, including Rab2, Rab4, Rab5, Rab8, Rab9, and Rab11. We demonstrate here a general and nearly invariant ability of GDI-2(WT) to release from membranes this subset of diverse Rabs. Deletion of an 18-residue segment from the C-terminal variable region yielded a fully functional or only slightly defective GDI-2. Conversely, substitution of Met at position 250 of the conserved region markedly abrogated the activity toward all Rabs. Surprisingly, a replacement of an adjacent conserved residue (Y249V) resulted in a selective Rab-dependent response and a profound gain of function toward specific Rabs. To further test whether the endogenous GDI-2 can adopt a gain-of-function conformation, we pharmacologically stimulated intact 3T3-L1 adipocytes to induce GDI-2 tyrosine phosphorylation. We found a pronounced increase of the Rab4 soluble form and its soluble complexes with the tyrosine-phosphorylated GDI-2. Together, these results indicate that (a) GDI-2 displays a general activity to release Rabs from membranes, (b) GDI-2-conserved residues, but not the C-terminal variable region, are essential for this activity, and (c) structural modifications in GDI-2 can enhance its functional activity, directing selective interactions with individual Rabs.     

Crucial roles of Rab22a in endosomal cargo recycling

Endosomal cargo recycling lies at the heart of subcellular trafficking processes under the management of several Ras-related GTP-binding proteins (Rabs) which are coordinated by their upstream regulators and require their downstream effectors to display their functions. In this regard, several Rabs have been well-reviewed except Rab22a. Rab22a is a crucial regulator of vesicle trafficking, early endosome and recycling endosome formation. Notably, recent studies demonstrated the immunological roles of Rab22a, which are closely associated with cancers, infection and autoimmune disorders. This review provides an overview of the regulators and effectors of Rab22a. Also, we highlight the current knowledge of the role of Rab22a in endosomal cargo recycling, including the biogenesis of recycling tubules with the help of a complex with Rab22a at its core, and how different internalized cargo chooses different recycling routes thanks to the cooperation of Rab22a, its effectors and its regulators. Of note, contradictions and speculation related to endosomal cargo recycling that Rab22a brings impacts on are also discussed. Finally, this review endeavors to briefly introduce the various events impacted by Rab22a, particularly focusing on the commandeered Rab22a-associated endosomal maturation and endosomal cargo recycling, in addition to the extensively investigated oncogenic role of Rab22a.     

Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes

Adipocytes hold the body's major energy reserve as triacylglycerols packaged in large lipid droplets. Perilipins, the most abundant proteins on these lipid droplets, play a critical role in facilitating both triacylglycerol storage and hydrolysis. The stimulation of lipolysis by beta-adrenergic agonists triggers rapid phosphorylation of perilipin and translocation of hormone-sensitive lipase to the surfaces of lipid droplets and more gradual fragmentation and dispersion of micro-lipid droplets. Because few lipid droplet-associated proteins have been identified in adipocytes, we isolated lipid droplets from basal and lipolytically stimulated 3T3-L1 adipocytes and identified the component proteins by mass spectrometry. Structural proteins identified in both preparations include perilipin, S3-12, vimentin, and TIP47; in contrast, adipophilin, caveolin-1, and tubulin selectively localized to droplets in lipolytically stimulated cells. Lipid metabolic enzymes identified in both preparations include hormone-sensitive lipase, lanosterol synthase, NAD(P)-dependent steroid dehydrogenase-like protein, acyl-CoA synthetase, long chain family member (ACSL) 1, and CGI-58. 17-beta-Hydroxysteroid dehydrogenase, type 7, was identified only in basal preparations, whereas ACSL3 and 4 and two short-chain reductase/dehydrogenases were identified on droplets from lipolytically stimulated cells. Additionally, both preparations contained FSP27, ribophorin I, EHD2, diaphorase I, and ancient ubiquitous protein. Basal preparations contained CGI-49, whereas lipid droplets from lipolytically stimulated cells contained several Rab GTPases and tumor protein D54. A close association of mitochondria with lipid droplets was suggested by the identification of pyruvate carboxylase, prohibitin, and a subunit of ATP synthase in the preparations. Thus, adipocyte lipid droplets contain specific structural proteins as well as lipid metabolic enzymes; the structural reorganization of lipid droplets in response to the hormonal stimulation of lipolysis is accompanied by increases in the relative mass of several proteins and the recruitment of additional proteins.     

Rabs 8A and 14 are targets of the insulin-regulated Rab-GAP AS160 regulating GLUT4 traffic in muscle cells

Insulin causes translocation of glucose transporter GLUT4 to the membrane of muscle and fat cells, a process requiring Akt activation. The Rab GTPase-activating protein (Rab-GAP) AS160 is inhibited upon phosphorylation by insulin-activated Akt, thereby allowing GLUT4 translocation. Although several Rab proteins are detected on GLUT4 vesicles, the target Rabs of AS160 involved in the GLUT4 translocation have not been identified. We test whether Rabs 8A, 10, and 14 (in vitro targets of AS160) rescue the inhibition of GLUT4 translocation caused by 'constitutively active' 4P-AS160 in L6 muscle cells. Coexpression of GFP-tagged Rabs 8A or Rab14 with 4P-AS160 prevented the inhibition of GLUT4 translocation imposed by 4P-AS160. GFP-tagged, constitutively active Rab8A also elicited this rescue. In contrast, neither wild-type nor constitutively active GFP-tagged Rab10 restored GLUT4 translocation. These results suggest that Rab8A and possibly Rab14 may be targets of AS160 leading to GLUT4 translocation in L6 muscle cells.     

TBC1D13 is a RAB35 Specific GAP that Plays an Important Role in GLUT4 Trafficking in Adipocytes

Insulin stimulates glucose transport in adipocytes by triggering translocation of GLUT4 glucose transporters to the plasma membrane (PM) and several Rabs including Rab10 have been implicated in this process. To delineate the molecular regulation of this pathway, we conducted a TBC/RabGAP overexpression screen in adipocytes. This identified TBC1D13 as a potent inhibitor of insulin-stimulated GLUT4 translocation without affecting other trafficking pathways. To determine the potential Rab substrate for TBC1D13 we conducted a yeast two-hybrid screen and found that the GTP bound forms of Rabs 1 and 10 specifically interacted with TBC1D13 but not with eight other TBC proteins. Surprisingly, a comprehensive in vitro screen for TBC1D13 GAP activity revealed Rab35 but not Rab10 as a specific substrate. TBC1D13 also displayed in vivo GAP activity towards Rab35. Overexpression of constitutively active Rab35 but not constitutively active Rab10 reversed the block in insulin-stimulated GLUT4 translocation observed with TBC1D13 overexpression. These studies implicate an important role for Rab35 in insulin-stimulated GLUT4 translocation in adipocytes.     

Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface

Rab proteins are a large family of monomeric GTPases with 60 members identified in the human genome. Rab GTPases require an isoprenyl modification to their C-terminus for membrane association and function in the regulation of vesicular trafficking pathways. This reaction is catalysed by Rab geranylgeranyl transferase, which recognises as protein substrate any given Rab in a 1:1 complex with Rab Escort Protein (REP). REP is therefore able to bind many distinct Rab proteins but the molecular basis for this activity is still unclear. We recently identified conserved motifs in Rabs termed RabF motifs, which we proposed to mediate a conserved mode of interaction between Rabs and REPs. Here, we tested this hypothesis. We first used REP1 as a bait in the yeast two-hybrid system and isolated strictly full-length Rabs, suggesting that REP recognises multiple regions within and properly folded Rabs. We introduced point mutations in Rab3a as a model Rab and assessed the ability of the mutants to interact with REP using the yeast two-hybrid system and an in vitro prenylation assay. We identified several residues that affect REP:Rab binding in the RabF1, RabF3, and RabF4 regions (which include parts of the switch I and II regions), but not other RabF regions. These results support the hypothesis that Rabs bind REP via conserved RabF motifs and provide a molecular explanation for the preferential recognition of the GDP-bound conformation of Rab by REP.     

Regulated localization of Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid droplet catabolism

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3DGV, a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3DGV labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.     

Thousands of rab GTPases for the cell biologist

Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology.     

Rab GTPases: specifying and deciphering organelle identity and function

Ten years ago, 20 Rab proteins had been identified as organelle-specific GTPases, and two were known to be essential for vesicle targeting in yeast. Today, more than 60 mammalian Rab proteins have been identified. While Rabs were always viewed as key regulatory factors, no one could have anticipated their diversity of functions and multitude of effectors. Rabs organize distinct protein scaffolds within a single organelle and act in a combinatorial manner with their effectors to regulate all stages of membrane traffic.