Rab GTPases Research Articles

Network and Evolutionary Analysis Reveals Candidate Genes of Membrane Trafficking Involved in Maize Seed Development and Immune Response

The plant membrane-trafficking system plays a crucial role in maintaining proper cellular functions and responding to various developmental and environmental cues. Thus far, our knowledge of the maize membrane-trafficking system is still limited. In this study, we systematically identified 479 membrane-trafficking genes from the maize genome using orthology search and studied their functions by integrating transcriptome and evolution analyses. These genes encode the components of coated vesicles, AP complexes, autophagy, ESCRTs, retromers, Rab GTPases, tethering factors, and SNAREs. The maize genes exhibited diverse but coordinated expression patterns, with 249 genes showing elevated expression in reproductive tissues. Further WGCNA analysis revealed that five COPII components and four Rab GTPases had high connectivity with protein biosynthesis during endosperm development and that eight components of autophagy, ESCRT, Rab, and SNARE were strongly co-upregulated with defense-related genes and/or with secondary metabolic processes to confer basal resistance to Fusarium graminearum. In addition, we identified 39 membrane-trafficking genes with strong selection signals during maize domestication and/or improvement. Among them, ZmSec23a and ZmVPS37A were selected for kernel oil production during improvement and pathogen resistance during domestication, respectively. In summary, these findings will provide important hints for future appreciation of the functions of membrane-trafficking genes in maize.

The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.

Potential role of Acanthamoeba Rab7

Acanthamoeba castellanii is a free-living protozoan that causes several severe human parasitic diseases such as Acanthamoeba keratitis and granulomatous encephalitis. A. castellanii feeds on bacteria, yeasts, and other organic particles as food sources, but the mechanisms of digestion in acanthamoebal cells are unclear. Rab GTPases participate in endosomal delivery in eukaryotes after phagocytosis. This study aimed to determine the potential functions of A. castellanii Rab7 (AcRab7), which is involved in phagocytosis, and the relationship between AcRab7 and further cellular physiological phenomena. In this study, the inhibitor CID1067700 (CID) was used to specifically inhibit the binding of nucleotides to confirm the potential functions of AcRab7. Cellular proliferation and ATP assays were also used to detect underlying cellular physiological functions after blocking the phagocytosis pathway. We found that AcRab7 expression increased as the co-culture time with Escherichia coli increased. Immunofluorescence staining showed that AcRab7 colocalized with lysosomes in its GTP-activating form. In addition, AcRab7 inhibition resulted in a reduction in cell proliferation and ATP levels. Our results suggest that AcRab7 participates in endosomal delivery and dominates energy production and cell growth.

Molecular characterization of Rab7 and its involvement in innate immunity in red swamp crayfish Procambarus clarkii

Rab7 is a member of the Rab GTPases protein family, and it plays an essential role in regulating trafficking organelles in higher animals. However, recent studies showed that it also participated in the immune response and cytophagy against pathogens in invertebrates. In the present study, the full-length of Rab7 from Procambarus clarkii (PcRab7) was cloned, and its function during pathogen infection and phagocytosis of haemocytes was also explored. The results showed that the full-length of PcRab7 was 3639 bp, containing a 618 bp open reading frame encoding 155 amino acids. The predicted molecular weight and isoelectric point of PcRab7 were 23.2 kDa and 5.77, respectively. PcRab7 was widely expressed in various tissues including haemocytes, intestine, muscle, gill, and hepatopancreas, and the highest expression level was in haemocytes. The mRNA transcripts of PcRab7 in the main organs (gill, intestine, and hepatopancreas, and haemocytes) were significantly affected by white spot syndrome virus (WSSV) and Aeromonas veronii infection. Subsequently, the prokaryotic and eukaryotic expression vectors were successfully constructed, and polyclonal antibodies, which could specifically recognize the endogenous Rab7 protein, were also obtained. Furthermore, the phagocytosis rate of haemocytes against FITC-labeled A. veronii was significantly decreased when the PcRab7 was silenced, while the over-expression of Rab7 increased the phagocytosis rate of haemocytes. The abnormal expression of Rab7 protein could also affect the survival rate of P. clarkii infected with WSSV or A. veronii. These results could provide a basis for further study on the immunological function of PcRab7.

Coronin1C Is a GDP-Specific Rab44 Effector That Controls Osteoclast Formation by Regulating Cell Motility in Macrophages.

Osteoclasts are multinucleated bone-resorbing cells that are formed by the fusion of macrophages. Recently, we identified Rab44, a large Rab GTPase, as an upregulated gene during osteoclast differentiation that negatively regulates osteoclast differentiation. However, the molecular mechanisms by which Rab44 negatively regulates osteoclast differentiation remain unknown. Here, we found that the GDP form of Rab44 interacted with the actin-binding protein, Coronin1C, in murine macrophages. Immunoprecipitation experiments revealed that the interaction of Rab44 and Coronin1C occurred in wild-type and a dominant-negative (DN) mutant of Rab44, but not in a constitutively active (CA) mutant of Rab44. Consistent with these findings, the expression of the CA mutant inhibited osteoclast differentiation, whereas that of the DN mutant enhanced this differentiation. Using a phase-contrast microscope, Coronin1C-knockdown osteoclasts apparently impaired multinuclear formation. Moreover, Coronin1C knockdown impaired the migration and chemotaxis of RAW-D macrophages. An in vivo experimental system demonstrated that Coronin1C knockdown suppresses osteoclastogenesis. Therefore, the decreased cell formation and fusion of Coronin1C-depleted osteoclasts might be due to the decreased migration of Coronin1C-knockdown macrophages. These results indicate that Coronin1C is a GDP-specific Rab44 effector that controls osteoclast formation by regulating cell motility in macrophages.

The regulatory role of the Aspergillus flavus core retromer complex in aflatoxin metabolism

Aflatoxins are a series of highly toxic and carcinogenic secondary metabolites that are synthesized by Aspergillus species. The degradation of aflatoxin enzymes is an important regulatory mechanism which modulates mycotoxin producing. The retromer complex is responsible for the retrograde transport of specific biomolecules and the vacuolar fusion in the intracellular transport. Late endosomal-associated GTPase (Rab7) has been shown to be a downstream effector protein of the retromer complex. A deficiency in the retromer complex or Rab7 results in several cellular trafficking problems in yeast and humans, like protein abnormal accumulation. However, whether retromer dysfunction is involved in aflatoxin synthesis remains unclear. Here, we report that the core retromer complex, which comprises three vacuolar protein sorting-associated proteins (AflVps26-AflVps29-AflVps35), is essential for the development of dormant and resistant fungal forms such as conidia (asexual reproductive spore) and sclerotia (hardened fungal mycelium), as well as aflatoxin production and pathogenicity, in Aspergillus flavus. In particular, we show the AflVps26-AflVps29-AflVps35 complex is negatively correlated with aflatoxin exportation. Structural simulation, site-specific mutagenesis, and coimmunoprecipitation experiments showed that interactions among AflVps26, AflVps29, and AflVps35 played crucial roles in the retromer complex executing its core functions. We further found an intrinsic connection between AflRab7 and the retromer involved in vesicle-vacuole fusion, which in turn affected the accumulation of aflatoxin synthesis-associated enzymes, suggesting that they work together to regulate the production of toxins. Overall, these results provide mechanistic insights that contribute to our understanding of the regulatory role of the core retromer complex in aflatoxin metabolism.

Distinct profiles of LRRK2 activation and Rab GTPase phosphorylation in clinical samples from different PD cohorts.

Despite several advances in the field, pharmacodynamic outcome measures reflective of LRRK2 kinase activity in clinical biofluids remain urgently needed. A variety of targets and approaches have been utilized including assessments of LRRK2 itself (levels, phosphorylation), or its substrates (e.g. Rab10 or other Rab GTPases). We have previously shown that intrinsic kinase activity of LRRK2 isolated from PBMCs of G2019S carriers is elevated, irrespective of disease status. In the present study we find that phosphorylation of Rab10 is also elevated in G2019S carriers, but only those with PD. Additionally, phosphorylation of this substrate is also elevated in two separate idiopathic PD cohorts, but not in carriers of the A53T mutation in α-synuclein. In contrast, Rab29 phosphorylation was specifically reduced in urinary exosomes from A53T and idiopathic PD patients. Taken together, our findings highlight the need for the assessment of multiple complimentary targets for a more comprehensive picture of the disease.

P53 Related Protein Kinase is Required for Arp2/3-Dependent Actin Dynamics of Hemocytes in Drosophila melanogaster.

Cells extend membrane protrusions like lamellipodia and filopodia from the leading edge to sense, to move and to form new contacts. The Arp2/3 complex sustains lamellipodia formation, and in conjunction with the actomyosin contractile system, provides mechanical strength to the cell. Drosophila p53-related protein kinase (Prpk), a Tsc5p ortholog, has been described as essential for cell growth and proliferation. In addition, Prpk interacts with proteins associated to actin filament dynamics such as α-spectrin and the Arp2/3 complex subunit Arpc4. Here, we investigated the role of Prpk in cell shape changes, specifically regarding actin filament dynamics and membrane protrusion formation. We found that reductions in Prpk alter cell shape and the structure of lamellipodia, mimicking the phenotypes evoked by Arp2/3 complex deficiencies. Prpk co-localize and co-immunoprecipitates with the Arp2/3 complex subunit Arpc1 and with the small GTPase Rab35. Importantly, expression of Rab35, known by its ability to recruit upstream regulators of the Arp2/3 complex, could rescue the Prpk knockdown phenotypes. Finally, we evaluated the requirement of Prpk in different developmental contexts, where it was shown to be essential for correct Arp2/3 complex distribution and actin dynamics required for hemocytes migration, recruitment, and phagocytosis during immune response.

Proteomics analysis of lipid droplets in mouse neuroblastoma cells

Lipid droplets (LDs) are intracellular droplets containing phospholipids and neutral lipids. It is well known that LDs are organelles with a rich proteome. In the nervous system, these droplets may play an important role in maintaining the normal physiological function of nerve cells. Moreover, LDs may relate to the neurodegenerative disorders, such as Alzheimer's disease (AD). However, more information is still needed about the function of LDs. In the study presented here, we identified the protein composition of mouse neuroblastoma (N2a) cell LDs using immunodetection and high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). Seventy three LDs proteins were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to investigate the potential functions of these proteins. Subsequently, the relationships among the proteins were analyzed by constructing a protein-protein interaction (PPI) network. N2a cell LDs contain multiple Rab GTPases, chaperones, and proteins involved in ubiquitination and transport. Some of these proteins were known to modulate LD formation and were related to the function of nerve cells. This work presents the proteome of N2a cell LDs and will help to identify the role of LDs in the nervous system.

Evidence for the role of Rab11-positive recycling endosomes as intermediates in coronavirus egress from epithelial cells

After their assembly by budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)–Golgi interface, coronaviruses (CoVs) are released from their host cells following a pathway that remains poorly understood. The traditional view that CoV exit occurs via the constitutive secretory route has recently been questioned by studies suggesting that this process involves unconventional secretion. Here, using the avian infectious bronchitis virus (IBV) as a well-established model virus, we have applied confocal microscopy to investigate the pathway of CoV egress from epithelial Vero cells. We report a novel effect of IBV infection on cellular endomembranes, namely, the compaction of the pericentrosomal endocytic recycling compartment (ERC) defined by the GTPase Rab11, which coincides with the previously described Golgi fragmentation, as well as virus release. Despite Golgi disassembly, the IC elements containing the major IBV membrane protein (M)—which mostly associates with newly formed virus particles—maintain their close spatial connection with the Rab11-positive endocytic recycling system. Moreover, partial colocalization of the M protein with Rab11 was observed, whereas M displayed negligible overlap with LAMP-1, indicating that IBV egress does not occur via late endosomes or lysosomes. Synchronization of virus release using temperature-shift protocols was accompanied by increased colocalization of M and Rab11 in vesicular and vacuolar structures in the pericentrosomal region and at the cell periphery, most likely representing IBV-containing transport carriers. In conclusion, these results add CoVs to the growing list of viruses exploiting the endocytic recycling apparatus defined by Rab11 for their assembly and/or release.

Therapeutic Targeting of Rab GTPases: Relevance for Alzheimer's Disease.

Rab GTPases (Rabs) are small proteins that play crucial roles in vesicle transport and membrane trafficking. Owing to their widespread functions in several steps of vesicle trafficking, Rabs have been implicated in the pathogenesis of several disorders, including cancer, diabetes, and multiple neurodegenerative diseases. As treatments for neurodegenerative conditions are currently rather limited, the identification and validation of novel therapeutic targets, such as Rabs, is of great importance. This review summarises proof-of-concept studies, demonstrating that modulation of Rab GTPases in the context of Alzheimer’s disease (AD) can ameliorate disease-related phenotypes, and provides an overview of the current state of the art for the pharmacological targeting of Rabs. Finally, we also discuss the barriers and challenges of therapeutically targeting these small proteins in humans, especially in the context of AD.

Alteration in Rab11-mediated endocytic trafficking of LDL receptor contributes to angiotensin II-induced cholesterol accumulation and injury in podocytes.

ObjectivesExposure of podocytes to angiotensin II (Ang II) enhances the abundance of the cell surface glycoprotein, low‐density lipoprotein receptor (LDLR) and promotes significant changes in the cellular cholesterol content. Recent investigation provides evidence that the small GTPase Rab11 is involved in the regulation of LDLR, but the exact mechanisms remain unknown. In this study, the role of Rab11 in post‐transcriptional regulation of LDLR was evaluated to investigate potential mechanisms of podocyte cholesterol dysregulation in chronic kidney disease.Materials and MethodsCholesterol content, LDLR and Rab11 expression were assessed in podocytes from Ang II‐infused mice. In vitro, the intracellular localization of LDLR was detected under different conditions. Rab11 expression was modulated and we then explored the effect of anti‐lipid cytotoxicity by detecting LDLR expression and trafficking, cholesterol content and apoptosis in podocytes.ResultsCholesterol accumulation, upregulated expression of LDLR and Rab11 were discovered in podocytes from Ang II‐infused mice. Ang II enhanced the co‐precipitation of LDLR with Rab11 and accelerated the endocytic recycling of LDLR to the plasma membrane. Additionally, silencing Rab11 promoted lysosomal degradation of LDLR and alleviated Ang II‐induced cholesterol accumulation and apoptosis in podocytes. Conversely, overexpression of Rab11 or inhibition of lysosomal degradation up‐regulated the abundance of LDLR and aggravated podocyte cholesterol deposition.ConclusionsRab11 triggers the endocytic trafficking and recycling of LDLR; overactivation of this pathway contributes to Ang II‐induced podocyte cholesterol accumulation and injury.

Rab39 and its effector UACA regulate basolateral exosome release from polarized epithelial cells.

Exosomes are small extracellular vesicles that originate from the intraluminal vesicles of multivesicular bodies (MVBs). We previously reported that polarized Madin-Darby canine kidney (MDCK) epithelial cells secrete two types of exosomes, apical and basolateral exosomes, from different MVBs. However, how these MVBs are selectively targeted to the apical or basolateral membrane remained unknown. Here, we analyze members of the Rab family small GTPases and show that different sets of Rabs mediate asymmetrical exosome release. Rab27, the best-known regulator of MVB transport for exosome release, is specifically but partially involved in apical exosome release, and Rab37, a close homolog of Rab27, is an additional apical exosome regulator. By contrast, Rab39 functions as a specific regulator of basolateral exosome release. Mechanistically, Rab39 interacts with its effector UACA, and UACA then recruits Lyspersin, a component of BLOC-1-related complex (BORC). Our findings suggest that the Rab39-UACA-BORC complex specifically mediates basolateral exosome release.

Transcriptional regulation of Rab21 GTPase in leukemia

Rab GTPase family proteins have critical roles in membrane trafficking. Rab21 is a member of the Ras oncogene family, whose dysregulation has been associated with drug resistance in cancer. Regulation of Rab21 expression is still unknown. Here we present data that RAB21 expression is regulated by Casein Kinase II (CK2) and by the Ikaros protein in B‐cell acute lymphoblastic leukemia (B‐ALL). Ikaros is a DNA‐binding protein, encoded by the Ikzf1 gene, which acts as a tumor suppressor in leukemia. Analysis of global DNA binding showed increased occupancy of Ikaros at promoter of the Rab21 gene in several B‐ALL cell lines and primary B‐ALL cells. The role of Ikaros in transcriptional regulation of RAB21 expression in B‐ALL was determined by gain‐of‐function and loss‐of‐function experiments. Overexpression of Ikaros in B‐ALL via retroviral transduction resulted in increased Ikaros binding to the RAB21 promoter and decreased RAB21 transcription. In contrast, shRNA‐mediated knockdown of Ikaros inhibited Ikaros binding to the RAB21 promoter and resulted in an increased RAB21 expression. These data suggest that Ikaros directly repress RAB21 transcription. Our previous studies showed that CK2 directly phosphorylates Ikaros and reduces its DNA‐binding affinity. We tested whether CK2 affects Ikaros’ ability to regulate RAB21 expression in B‐ALL. Treatment of B‐ALL cells with a specific CK2 inhibitors, CX‐4945, resulted in increased Ikaros binding at the RAB21 promoter and reduced RAB21 expression. The use of luciferase reporter assay showed that both wildtype Ikaros and Ikaros phosphoresistant mutants strongly repress Rab21 transcription, while phopshomimetic Ikaros mutants at CK2 phosphorylation sites lost the ability to repress RAB21 transcription. Together, these data demonstrate that Ikaros and CK2 regulate transcription and overall expression of RAB21 in B‐cell ALL. Results uncovered an interplay between CK2 and Rab GTPase signaling pathways and suggest a mechanism by which CK2 regulates Rab21 activity in leukemia.

Structural basis for Rab6 GTPase activation by the Ric1‐Rgp1 complex

Membrane trafficking is a highly coordinated process in eukaryotes essential for maintaining lipid homeostasis and sorting proteins to the proper location inside cells. At the hub of this dynamic set of pathways, the Golgi complex directs much of this sorting through vesicle mediated transport. Vesicles of varying lipid composition loaded with different protein cargo are in constant flux through the Golgi and precise coordination of these processes requires communication between compartments. Rab GTPases are small signaling proteins that act as molecular switches to regulate vesicular trafficking. Rab6 functions at the Golgi and is important for endosome to Golgi retrograde transport in both yeast and metazoans and for anterograde exit from the TGN in metazoans. The Ric1‐Rgp1 protein complex is a highly conserved guanine nucleotide exchange factor (GEF) required for activation of Rab6. In mammalian cells Ric1‐Rgp1 is known to be regulated by the Rab33 GTPase, but it remains unresolved how the activity of the complex is regulated and how it interacts with Rab6. Furthermore, very little is known about the structure and domain organization of Ric1‐Rgp1. We have used cryo‐EM to determine the high‐resolution structure of the yeast Ric1‐Rgp1‐Rab6(Ypt6) complex, representing the key intermediate of the nucleotide exchange reaction. This structure reveals the overall architecture of the complex and has enabled us to identify the specific interactions that govern activation of Rab6. Ongoing studies aim to test the physiological significance of these interactions and determine the orientation of this complex on the Golgi membrane surface.

Auto‐inhibitory interactions of Sec7, master regulator of the Golgi complex

In eukaryotes, protein and lipid secretory trafficking is orchestrated by the Golgi apparatus where proteins are modified and sorted in a cascade of functionally distinct compartments. The composition of these compartments is maintained primarily by vesicular traffic facilitated by Arf and Rab GTPases. These GTPases are regulated by a GTP/GDP cycle facilitated by Guanine Nucleotide Exchange factors (GEFs) and GTPase Activating Proteins (GAPs) to induce GDP/GTP exchange and GTP hydrolysis respectively. Vesicular trafficking to and from the final compartment, the trans‐Golgi network, maintains membrane flux throughout the cell with cargo recycling and secretory pathways. Arf1 and its close paralogs are responsible for virtually all vesicle biogenesis events at the Golgi complex. In budding yeast, Arf1 is activated at the trans‐Golgi network by the Arf GEF Sec7, the homolog of the human ARFGEF/BIG proteins. Previous work has determined that Sec7 is regulated by autoinhibition, positive feedback, and GTPase crosstalk, yet the mechanistic basis for these regulatory features is unknown. To understand how Sec7 is regulated, I have determined the structure of the flexible Sec7 dimer by cryoEM at 4.9A resolution. Focused refinements on individual monomer subunits improved resolution to 3.8A, sufficient for molecular model building. This structure reveals the mechanism for Sec7 autoinhibition: in the cytosol, the GEF domain interacts with the HDS domains in a manner that prevents binding to the Arf1 substrate. We also observe that the dimerization region imparts a twist between the two monomers, apparently preventing both catalytic subunits from interacting with the membrane surface simultaneously. Functional studies are currently underway to determine the physiological relevance of our structural findings.

Identifying Vesicular Localization of the Rho GTPase RhoJ/TCL Through Colocalization with Rab Family GTPases

TCL belongs to the Rho family of GTPases and contributes to tumor‐associated angiogenesis. Understanding its basic cell biology is essential for establishing its role in this process. Experiments in several labs have shown plasma membrane and vesicular localizations of TCL, but it is less clear as to what subtype of vesicle the GTPase associates with and how it may influence vesicle trafficking. To better understand this aspect of TCL cellular biology, a YFP‐tagged version of TCL was co‐expressed in HeLa cells with sixteen different mCherry‐tagged versions of Rab GTPases. Images were captured using an Olympus Fluoview confocal microscope and colocalization assessed both qualitatively and quantitatively using CellSens software. CL colocalized with Rab5 and 7 as previously published but we also show colocalization with Rab17 and 40. Since Rab5, 7, and 17 Rab GTPases have reported vesicular localizations and functions, we intend to assess whether TCL plays any roles in their established pathways.

Microautophagy catabolizes lipid droplets in liver hepatocytes via the small GTPase Rab5 and is connected to alcoholic fatty liver

Introduction and ObjectiveAlcohol consumption has increased dramatically in recent years, representing a significant challenge to human health in metabolic disease. The leading cause of alcohol‐related death is alcohol‐associated liver disease (AALD), which is present at early stages (i.e. steatosis) in 90% of heavy drinkers. During early AALD, liver hepatocytes accumulate large stores of triglycerides in the form of lipid droplets (LDs). Under normal conditions, degradative lysosomes form direct contacts with LDs and catabolize lipid via microlipophagy, a specialized autophagic pathway utilizing the lipid‐degrading enzyme lysosomal acid lipase (LAL). However, the trafficking mechanisms that govern LD‐lysosome contacts during microlipophagy are poorly understood. Several promising candidates exist within the Rab family of small GTPases, which are well‐known to direct the trafficking of intracellular vesicles and LDs. Moreover, previous studies suggest that Rab proteins play central roles in LD catabolism. Thus, the OBJECTIVE of this study was to investigate novel Rab proteins that might be impaired by EtOH leading to altered LD dynamics and microautophagy.Materials and MethodsTo this end, we utilized RNAi gene silencing, subcellular fractionation, Western blot analysis, and high‐resolution microscopy to define Rabs that might target LDs to lysosomes during microlipophagy. To mimic alcoholic fatty liver, cellular and animal models of alcohol consumption were also used. Changes in LD‐lysosome interaction were measured by quantitative confocal microscopy following pharmacological inhibition of LAL by LAListat.ResultsFirst, confocal microscopy revealed that GFP‐Rab5, an early endosomal Rab GTPase, localizes to hepatocellular LDs consistent with proteomic and Western blot analyses showing abundant levels of Rab5 in biochemically isolated LDs. Importantly, siRNA knockdown of Rab5 in cultured hepatocytes showed substantial increases in total LD area (2‐fold) and LD number (1.6‐fold), and also blocked the catabolism of LDs in lipid‐loaded cells. Finally, Rab5 knockdown nearly completely abolished microlipophagy as measured by fluorescent LD accumulation in lysosomes following LAListat treatment. The dissociation of LDs from lysosomes in Rab5 knockdown cells was strikingly similar to the defect observed in EtOH‐fed hepatocytes. Together, these findings suggest that Rab5 is a key mediator of targeting LDs to the lysosome compartment for catabolism and may be a target of EtOH insult.Significance/ImplicationThis study sheds light on new molecular mechanisms of microlipophagy that are affected by chronic alcohol consumption. These mechanisms may represent promising avenues for future therapeutic development toward mitigating hepatic steatosis and slowing the progression of AALD.

Structural basis for specific activation of the Rab11 GTPase by the TRAPPII complex

Secretion is an essential cellular function in which proteins and lipids are transported from the Golgi to the plasma membrane via secretory vesicles. The formation of secretory vesicles is regulated by activation of the Rab GTPase Rab11. The activation of Rab GTPases at membrane compartments is dependent on the localization of their specific Guanosine Exchange Factors (GEFs), which catalyze the exchange of GDP with GTP and stabilize the active GTPase on the membrane. The multisubunit TRAPPII complex acts as Rab11 GEF and recruits it to the trans‐Golgi network (TGN) membrane. More than 60 different Rabs are present in mammals and budding yeast has 11 different Rabs. Specific localization of each of these Rabs to different membrane compartments is important for determination of organelle identity and membrane organization of these compartments. While multiple GTPases and GEFs localize at the TGN, the mechanistic details of how TRAPPII specifically activates Rab11, and not other GTPases, is unknown. Moreover, another related GEF, the TRAPPIII complex, shares the same catalytic subunits with TRAPPII and yet activates another GTPase, Rab1. A steric gating mechanism has been proposed in which TRAPPII selects against Rab1 based on the length of the C‐terminal hypervariable domain. We determined the structure of yeast TRAPPII bound to its substrate Rab11 using cryo‐EM at an overall resolution of 3.7Å. Analysis of the atomic structure has revealed specific interactions between Rab11 and the TRAPPII complex, and how TRAPPII interacts with the membrane surface. We tested the physiological relevance of these observed interactions using in vivo functional studies and in vitro reconstitution of nucleotide exchange. We show that the TRAPPII subunit Trs130 provides a ‘leg’ which lifts the TRAPP catalytic site above the membrane, thereby preventing access to the catalytic site by Rab1. TRAPPIII complex selects against Rab11 based on repulsive interactions with the catalytic subunits. We also show an alternative conformation of the TRAPPII complex which may facilitate access of Rab11 to the TRAPP catalytic site. Taken together, these experiments reveal the mechanism of specific activation of Rab11 on the membrane by TRAPPII, a key step for initiation of secretory vesicle formation. As Rab11 and the TRAPPII complex are conserved throughout eukaryotes, this mechanism may be widely conserved across all eukaryotic secretory systems.

RAB GTPases and SNAREs at the trans-Golgi network in plants

Membrane traffic is a fundamental cellular system to exchange proteins and membrane lipids among single membrane-bound organelles or between an organelle and the plasma membrane in order to keep integrity of the endomembrane system. RAB GTPases and SNARE proteins, the key regulators of membrane traffic, are conserved broadly among eukaryotic species. However, genome-wide analyses showed that organization of RABs and SNAREs that regulate the post-Golgi transport pathways is greatly diversified in plants compared to other model eukaryotes. Furthermore, some organelles acquired unique properties in plant lineages. Like in other eukaryotic systems, the trans-Golgi network of plants coordinates secretion and vacuolar transport; however, uniquely in plants, it also acts as a platform for endocytic transport and recycling. In this review, we focus on RAB GTPases and SNAREs that function at the TGN, and summarize how these regulators perform to control different transport pathways at the plant TGN. We also highlight the current knowledge of RABs and SNAREs’ role in regulation of plant development and plant responses to environmental stimuli.