The Vps13 Family of Lipid Transporters and Its Role at Membrane Contact Sites

The conserved VPS13 proteins constitute a new family of lipid transporters at membrane contact sites. These large proteins are suspected to bridge membranes and form a direct channel for lipid transport between organelles. Mutations in the 4 human homologs (VPS13A–D) are associated with a number of neurological disorders, but little is known about their precise functions or the relevant contact sites affected in disease. In contrast, yeast has a single Vps13 protein which is recruited to multiple organelles and contact sites. The yeast model system has proved useful for studying the function of Vps13 at different organelles and identifying the localization determinants responsible for its membrane targeting. In this review we describe recent advances in our understanding of VPS13 proteins with a focus on yeast research.     

Genetic Dissection of Vps13 Regulation in Yeast Using Disease Mutations from Human Orthologs

The VPS13 family of proteins have emerged as key players in intracellular lipid transport and human health. Humans have four different VPS13 orthologs, the dysfunction of which leads to different diseases. Yeast has a single VPS13 gene, which encodes a protein that localizes to multiple different membrane contact sites. The yeast vps13Δ mutant is pleiotropic, exhibiting defects in sporulation, protein trafficking, endoplasmic reticulum (ER)-phagy and mitochondrial function. Non-null alleles resulting from missense mutations can be useful reagents for understanding the multiple functions of a gene. The exceptionally large size of Vps13 makes the identification of key residues challenging. As a means to identify critical residues in yeast Vps13, amino acid substitution mutations from VPS13A, B, C and D, associated with human disease, were introduced at the cognate positions of yeast VPS13, some of which created separation-of-function alleles. Phenotypic analyses of these mutants have revealed that the promotion of ER-phagy is a fourth, genetically separable role of VPS13 and provide evidence that co-adaptors at the endosome mediate the activity of VPS13 in vacuolar sorting.     

The p24 Complex Contributes to Specify Arf1 for COPI Coat Selection

Golgi trafficking depends on the small GTPase Arf1 which, upon activation, drives the assembly of different coats onto budding vesicles. Two related types of guanine nucleotide exchange factors (GEFs) activate Arf1 at different Golgi sites. In yeast, Gea1 in the cis-Golgi and Gea2 in the medial-Golgi activate Arf1 to form COPI­coated vesicles for retrograde cargo sorting, whereas Sec7 generates clathrin/adaptor­coated vesicles at the trans-Golgi network (TGN) for forward cargo transport. A central question is how the same activated Arf1 protein manages to assemble different coats depending on the donor Golgi compartment. A previous study has postulated that the interaction between Gea1 and COPI would channel Arf1 activation for COPI vesicle budding. Here, we found that the p24 complex, a major COPI vesicle cargo, promotes the binding of Gea1 with COPI by increasing the COPI association to the membrane independently of Arf1 activation. Furthermore, the p24 complex also facilitates the interaction of Arf1 with its COPI effector. Therefore, our study supports a mechanism by which the p24 complex contributes to program Arf1 activation by Gea1 for selective COPI coat assembly at the cis-Golgi compartment.     

Targeting Copper Homeostasis Improves Functioning of vps13Δ Yeast Mutant Cells,a Model of VPS13-Related Diseases

Ion homeostasis is crucial for organism functioning, and its alterations may cause diseases. For example, copper insufficiency and overload are associated with Menkes and Wilson’s diseases, respectively, and iron imbalance is observed in Parkinson’s and Alzheimer’s diseases. To better understand human diseases, Saccharomyces cerevisiae yeast are used as a model organism. In our studies, we used the vps13Δ yeast strain as a model of rare neurological diseases caused by mutations in VPS13A–D genes. In this work, we show that overexpression of genes encoding copper transporters, CTR1, CTR3, and CCC2, or the addition of copper salt to the medium, improved functioning of the vps13Δ mutant. We show that their mechanism of action, at least partially, depends on increasing iron content in the cells by the copper-dependent iron uptake system. Finally, we present that treatment with copper ionophores, disulfiram, elesclomol, and sodium pyrithione, also resulted in alleviation of the defects observed in vps13Δ cells. Our study points at copper and iron homeostasis as a potential therapeutic target for further investigation in higher eukaryotic models of VPS13-related diseases.     

Expression of Low Level of VPS35-mCherry Fusion Protein Diminishes Vps35 Depletion Induced Neuron Terminal Differentiation Deficits and Neurodegenerative Pathology,and Prevents Neonatal Death

Vps35 (vacuolar protein sorting 35) is a key component of retromer that consists of Vps35, Vps26, and Vps29 trimers, and sortin nexin dimers. Dysfunctional Vps35/retromer is believed to be a risk factor for development of various neurodegenerative diseases. Vps35^Neurod6 mice, which selectively knock out Vps35 in Neurod6-Cre+ pyramidal neurons, exhibit age-dependent impairments in terminal differentiation of dendrites and axons of cortical and hippocampal neurons, neuro-degenerative pathology (i.e., increases in P62 and Tdp43 (TAR DNA-binding protein 43) proteins, cell death, and reactive gliosis), and neonatal death. The relationships among these phenotypes and the underlying mechanisms remain largely unclear. Here, we provide evidence that expression of low level of VPS35-mCherry fusion protein in Vps35^Neurod6 mice could diminish the phenotypes in an age-dependent manner. Specifically, we have generated a conditional transgenic mouse line, LSL-Vps35-mCherry, which expresses VPS35-mCherry fusion protein in a Cre-dependent manner. Crossing LSL-Vps35-mCherry with Vps35^Neurod6 to obtain TgVPS35-mCherry, Vps35^Neurod6 mice prevent the neonatal death and diminish the dendritic morphogenesis deficit and gliosis at the neonatal, but not the adult age. Further studies revealed that the Vps35-mCherry transgene expression was low, and the level of Vps35 mRNA comprised only ~5–7% of the Vps35 mRNA of control mice. Such low level of VPS35-mCherry could restore the amount of other retromer components (Vps26a and Vps29) at the neonatal age (P14). Importantly, the neurodegenerative pathology presented in the survived adult TgVps35-mCherry; Vps35^Neurod6 mice. These results demonstrate the sufficiency of low level of VPS35-mCherry fusion protein to diminish the phenotypes in Vps35^Neurod6 mice at the neonatal age, verifying a key role of neuronal Vps35 in stabilizing retromer complex proteins, and supporting the view for Vps35 as a potential therapeutic target for neurodegenerative diseases.     

Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model

The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM), or the external medium, via the exocytosis or secretory pathway (SEC), and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway) or directly (alkaline phosphatase or ALP pathway). Plasma membrane proteins can be internalized by endocytosis (END) and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway). Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes.     

Small GTPases of the Rab and Arf Families: Key Regulators of Intracellular Trafficking in Neurodegeneration

Small guanosine triphosphatases (GTPases) of the Rab and Arf families are key regulators of vesicle formation and membrane trafficking. Membrane transport plays an important role in the central nervous system. In this regard, neurons require a constant flow of membranes for the correct distribution of receptors, for the precise composition of proteins and organelles in dendrites and axons, for the continuous exocytosis/endocytosis of synaptic vesicles and for the elimination of dysfunctional proteins. Thus, it is not surprising that Rab and Arf GTPases have been associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Both pathologies share characteristics such as the presence of protein aggregates and/or the fragmentation of the Golgi apparatus, hallmarks that have been related to both Rab and Arf GTPases functions. Despite their relationship with neurodegenerative disorders, very few studies have focused on the role of these GTPases in the pathogenesis of neurodegeneration. In this review, we summarize their importance in the onset and progression of Alzheimer’s and Parkinson’s diseases, as well as their emergence as potential therapeutical targets for neurodegeneration.     

Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit

Auxin response factors (ARFs) play important roles in various plant physiological processes; however, knowledge of the exact role of ARFs in plant responses to water deficit is limited. In this study, SlARF4, a member of the ARF family, was functionally characterized under water deficit. Real-time fluorescence quantitative polymerase chain reaction (PCR) and β-glucuronidase (GUS) staining showed that water deficit and abscisic acid (ABA) treatment reduced the expression of SlARF4. SlARF4 was expressed in the vascular bundles and guard cells of tomato stomata. Loss of function of SlARF4 (arf4) by using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 (CRISPR/Cas 9) technology enhanced plant resistance to water stress and rehydration ability. The arf4 mutant plants exhibited curly leaves and a thick stem. Malondialdehyde content was significantly lower in arf4 mutants than in wildtype plants under water stress; furthermore, arf4 mutants showed higher content of antioxidant substances, superoxide dismutase, actual photochemical efficiency of photosystem II (PSII), and catalase activities. Stomatal and vascular bundle morphology was changed in arf4 mutants. We identified 628 differentially expressed genes specifically expressed under water deficit in arf4 mutants; six of these genes, including ABA signaling pathway-related genes, were differentially expressed between the wildtype and arf4 mutants under water deficit and unlimited water supply. Auxin responsive element (AuxRE) elements were found in these genes’ promoters indicating that SlARF4 participates in ABA signaling pathways by regulating the expression of SlABI5/ABF and SCL3, thereby influencing stomatal morphology and vascular bundle development and ultimately improving plant resistance to water deficit.     

A Genome-Wide Screen Reveals That Endocytic Genes Are Important for Pma1p Asymmetry during Cell Division in Saccharomyces cerevisiae

An asymmetry in cytosolic pH between mother and daughter cells was reported to underlie cellular aging in the budding yeast Saccharomyces cerevisiae; however, the underlying mechanism remains unknown. Preferential accumulation of Pma1p, which pumps cytoplasmic protons out of cells, at the plasma membrane of mother cells, but not of their newly-formed daughter cells, is believed to be responsible for the pH increase in mother cells by reducing the level of cytoplasmic protons. This, in turn, decreases the acidity of vacuoles, which is well correlated with aging of yeast cells. In this study, to identify genes that regulate the preferential accumulation of Pma1p in mother cells, we performed a genome-wide screen using a collection of single gene deletion yeast strains. A subset of genes involved in the endocytic pathway, such as VPS8, VPS9, and VPS21, was important for Pma1p accumulation. Unexpectedly, however, there was little correlation between deletion of each of these genes and the replicative lifespan of yeast, suggesting that Pma1p accumulation in mother cells is not the key determinant that underlies aging of mother cells.     

Lack of GABARAP-Type Proteins Is Accompanied by Altered Golgi Morphology and Surfaceome Composition

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome composition of TKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.     

TFIP11 Interacts with mDEAH9, an RNA Helicase Involved in Spliceosome Disassembly

Yeast proteins Ntr1, Ntr2 and Prp43 function in spliceosome disassembly. An Ntr1-Ntr2 protein complex recruits Prp43 to allow the removal of the lariat-intron in late-stage RNA splicing activity. Based on amino-acid sequence similarities across species, TFIP11 and mDEAH9/Dhx15 have been identified as homologues of yeast Ntr1 and Prp43, respectively. The N-terminal region of TFIP11 contains a G-patch, which is a highly conserved domain of many RNA-processing proteins. TFIP11 displays a unique and characteristic subnuclear localization pattern, in close proximity to SC35 nuclear speckles. Transfected GFP-tagged mDEAH9 displays an evenly distributed nuclear localization and is excluded from the nucleoli; however when TFIP11 and mDEAH9 are co-transfected, both proteins colocalize to distinct nuclear speckles. These data show that TFIP11 recruits mDEAH9 suggesting that these two proteins have similar biological activities to their yeast counterparts.     

Aedes aegypti Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization

The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from Drosophila studies to maintain the integrity of the germline genome by silencing transposable elements (TE). Aedes aegypti mosquitoes, which are the key vectors of several arthropod-borne viruses, exhibit an expanded repertoire of Piwi proteins involved in the piRNA pathway, suggesting functional divergence. Here, we investigate RNA-binding dynamics and subcellular localization of A. aegypti Piwi4 (AePiwi4), a Piwi protein involved in antiviral immunity and embryonic development, to better understand its function. We found that AePiwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3′ ends of piRNAs, bound to mature (3′ 2′ O-methylated) and unmethylated RNAs with similar micromolar affinities (K_D = 1.7 ± 0.8 μM and K_D of 5.0 ± 2.2 μM, respectively; p = 0.05) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins have significant impacts on binding behaviors, likely by impacting the protein secondary structure. We also analyzed AePiwi4 subcellular localization in mosquito tissues. We found that the protein is both cytoplasmic and nuclear, and we identified an AePiwi4 nuclear localization signal (NLS) in the N-terminal region of the protein. Taken together, these studies provide insights on the dynamic role of AePiwi4 in RNAi and pave the way for future studies aimed at understanding Piwi interactions with diverse RNA populations.     

Molecular Characterization of U-box E3 Ubiquitin Ligases (TaPUB2 and TaPUB3) Involved in the Positive Regulation of Drought Stress Response in Arabidopsis

Plant U-box E3 ubiquitin ligase (PUB) is involved in various environmental stress conditions. However, the molecular mechanism of U-box proteins in response to abiotic stress in wheat remains unknown. In this study, two U-box E3 ligase genes (TaPUB2 and TaPUB3), which are highly expressed in response to adverse abiotic stresses, were isolated from common wheat, and their cellular functions were characterized under drought stress. Transient expression assay revealed that TaPUB2 was localized in the cytoplasm and Golgi apparatus, whereas TaPUB3 was expressed only in the Golgi apparatus in wheat protoplasts. Additionally, TaPUB2 and TaPUB3 underwent self-ubiquitination. Moreover, TaPUB2/TaPUB3 heterodimer was identified in yeast and the cytoplasm of wheat protoplasts using a pull-down assay and bimolecular fluorescence complementation analysis. Heterogeneous overexpression of TaPUB2 and TaPUB3 conferred tolerance to drought stress. Taken together, these results implied that the heterodimeric form of U-box E3 ubiquitin ligases (TaPUB2/TaPUB3) responded to abiotic stress and roles as a positive regulator of drought stress tolerance.     

The Intracellular Distribution of the Small GTPase Rho5 and Its Dimeric Guanidine Nucleotide Exchange Factor Dck1/Lmo1 Determine Their Function in Oxidative Stress Response

Rho5, the yeast homolog of human Rac1, is a small GTPase which regulates the cell response to nutrient and oxidative stress by inducing mitophagy and apoptosis. It is activated by a dimeric GEF composed of the subunits Dck1 and Lmo1. Upon stress, all three proteins rapidly translocate from the cell surface (Rho5) and a diffuse cytosolic distribution (Dck1 and Lmo1) to mitochondria, with translocation of the GTPase depending on both GEF subunits. We here show that the latter associate with mitochondria independent from each other and from Rho5. The trapping of Dck1-GFP or GFP-Lmo1 to the mitochondrial surface by a specific nanobody fused to the transmembrane domain (TMD) of Fis1 results in a loss of function, mimicking the phenotypes of the respective gene deletions, dck1 or lmo1. Direct fusion of Rho5 to Fis1^TMD, i.e., permanent attachment to the mitochondria, also mimics the phenotypes of an rho5 deletion. Together, these data suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation in order to fulfill its function in the oxidative stress response. This notion is substantiated by the observation that strains carrying fusions of Rho5 to the cell wall integrity sensor Mid2, confining the GTPase to the plasma membrane, retained their function. We propose a model in which Rho5 activated at the plasma membrane represses the oxidative stress response under standard growth conditions. This repression is relieved upon its GEF-mediated translocation to mitochondria, thus triggering mitophagy and apoptosis.     

Novel Insights into Selected Disease-Causing Mutations within the SLC35A1 Gene Encoding the CMP-Sialic Acid Transporter

Congenital disorders of glycosylation (CDG) are a group of rare genetic and metabolic diseases caused by alterations in glycosylation pathways. Five patients bearing CDG-causing mutations in the SLC35A1 gene encoding the CMP-sialic acid transporter (CST) have been reported to date. In this study we examined how specific mutations in the SLC35A1 gene affect the protein’s properties in two previously described SLC35A1-CDG cases: one caused by a substitution (Q101H) and another involving a compound heterozygous mutation (T156R/E196K). The effects of single mutations and the combination of T156R and E196K mutations on the CST’s functionality was examined separately in CST-deficient HEK293T cells. As shown by microscopic studies, none of the CDG-causing mutations affected the protein’s proper localization in the Golgi apparatus. Cellular glycophenotypes were characterized using lectins, structural assignment of N- and O-glycans and analysis of glycolipids. Single Q101H, T156R and E196K mutants were able to partially restore sialylation in CST-deficient cells, and the deleterious effect of a single T156R or E196K mutation on the CST functionality was strongly enhanced upon their combination. We also revealed differences in the ability of CST variants to form dimers. The results of this study improve our understanding of the molecular background of SLC35A1-CDG cases.     

IKKγ/NEMO Localization into Multivesicular Bodies

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3β (GSK-3β) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3β leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3β and V-ATPase in NF-κB signaling activation.     

Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

Golgin-160 is a member of the golgin family of proteins, which have been implicated in the maintenance of Golgi structure and in vesicle tethering. Golgin-160 is atypical; it promotes post-Golgi trafficking of specific cargo proteins, including the β-1 adrenergic receptor (β1AR), a G protein-coupled receptor. Here we show that golgin-160 binds directly to the third intracellular loop of β1AR and that this binding depends on three basic residues in this loop. Mutation of the basic residues does not affect trafficking of β1AR from the endoplasmic reticulum through the Golgi complex, but results in reduced steady-state levels at the plasma membrane. We hypothesize that golgin-160 promotes incorporation of β1AR into specific transport carriers at the trans-Golgi network to ensure efficient delivery to the cell surface. These results add to our understanding of the biogenesis of β1AR, and suggest a novel point of regulation for its delivery to the plasma membrane.     

HPE1, an Effector from Zebra Chip Pathogen Interacts with Tomato Proteins and Perturbs Ubiquitinated Protein Accumulation

The gram-negative bacterial genus Liberibacter includes economically important pathogens, such as ‘Candidatus Liberibacter asiaticus’ that cause citrus greening disease (or Huanglongbing, HLB) and ‘Ca. Liberibacter solanacearum’ (Lso) that cause zebra chip disease in potato. Liberibacter pathogens are fastidious bacteria transmitted by psyllids. Pathogen manipulation of the host’ and vector’s immune system for successful colonization is hypothesized to be achieved by Sec translocon-dependent effectors (SDE). In previous work, we identified hypothetical protein effector 1 (HPE1), an SDE from Lso, that acts as a suppressor of the plant’s effector-triggered immunity (ETI)-like response. In this study, using a yeast two-hybrid system, we identify binding interactions between tomato RAD23 proteins and HPE1. We further show that HPE1 interacts with RAD23 in both nuclear and cytoplasmic compartments in planta. Immunoblot assays show that HPE1 is not ubiquitinated in the plant cell, but rather the expression of HPE1 induced the accumulation of other ubiquitinated proteins. A similar accumulation of ubiquitinated proteins is also observed in Lso infected tomato plants. Finally, earlier colonization and symptom development following Lso haplotype B infection are observed in HPE1 overexpressing plants compared to wild-type plants. Overall, our results suggest that HPE1 plays a role in virulence in Lso pathogenesis, possibly by perturbing the ubiquitin-proteasome system via direct interaction with the ubiquitin-like domain of RAD23 proteins.