47 research outputs found
Identification and functional analysis of the endosomal VINE complex in Saccharomyces cerevisiae
No full textThe endosome is a central sorting station for proteins and lipids. Retrograde protein sorting from the endosome is promoted by conserved SNX-BAR-containing coat complexes including retromer which enrich cargo at tubular microdomains and generate transport carriers. In humans, retromer cooperates with the VPS9-domain GEF VARP to direct an endosomal recycling pathway. Functions of the yeast VARP homolog Vrl1 have been overlooked due to an inactivating mutation in commonly studied strains. It is not clear how Vrl1 contributes to endosomal sorting and investigating this could provide insight to pathways controlled by VARP. Here, we show that Vrl1 is an endosomal protein with features of a SNX-BAR. We demonstrate that Vrl1 associates with Vin1, the paralog of retromer SNX-BAR Vps5, and that this is essential for its localization and function. Unique features in the Vin1 N-terminus allow Vrl1 to distinguish it from Vps5, thereby forming a complex that we have named VINE. Like other SNX-BAR coats, VINE localizes to endosomes in a PI3P-dependent manner and templates endosomal tubules, indicating a potential role in membrane trafficking. Vin1 and Vps5 specifically form VINE and retromer, respectively, through interactions at their shared unstructured N-terminal regions. We mapped determinants of VINE formation in the Vin1 N-terminus and identified a poly-basic region and a Leu-Phe motif. Structural prediction of the Vps5 N-terminal interaction with retromer subunit Vps29 suggest a bipartite mechanism which also involves a Leu-Phe motif. By establishing an ectopic expression system in S. cerevisiae, we demonstrate that the sole isoform of Vps5 in closely related yeast K. lactis forms both VINE and retromer. Structural predictions of the K. lactis Vps5 N-terminus suggest a regulated or competitive mechanism could control complex selection between VINE and retromer. In this work we identified and characterized mechanisms of assembly for a novel yeast VPS9 domain-containing SNX-BAR coat that we have named VINE. VINE combines features of a sorting complex and GEF, and may perform roles in endosomal membrane trafficking. The findings outlined in this dissertation advance our understanding of SNX-BAR assembly in yeast and humans and provide insight to the functional relationship between endosomal coats and VPS9-domain GEFs.Medicine, Faculty ofMedical Genetics, Department ofGraduat
Investigating the role of the putative lipid transport protein Fmp27 at ER-PM membrane contact sites
No full textEukaryotic organelle membranes are composed of lipids and proteins. Most lipid precursors are synthesized in the endoplasmic reticulum (ER) and are transported to other membranes. Transport can proceed either via the vesicular or the non-vesicular route, primarily via lipid transport proteins at membrane contact sites (MCSs). MCSs are sites of close apposition between organelle membranes, which are present in eukaryotes including plants, fungi and animals. These sites are maintained by protein tethers, which function in lipid transport, maintenance of membrane integrity, membrane biogenesis and ion regulation. One such protein tether, Vps13, is highly conserved and functions as a bulk lipid transporter at many MCSs. Bulk transport of lipids is required to confer rapid changes in lipid composition or for membrane elongation, such as in case of generation of prospore membranes or for autophagosome formation or during endocytosis. At ER-PM MCSs, Vps13-like proteins could function in membrane elongation during endocytosis, thus making it important to identify Vps13-like proteins at these sites. I identified an uncharacterized protein Fmp27 and its paralog Ypr117w, which have secondary structure similarities to Vps13, with a potential role in lipid homeostasis at ER-PM MCSs. Fmp27 is a well conserved protein, and its mammalian ortholog is overexpressed in breast cancer, whereas its fly and plant orthologs are implicated in organismal development. I found that Fmp27 is anchored to the ER and localizes to ER-PM cortical sites. Furthermore, I identified an interacting partner, Ybl086c, which binds to Fmp27 and recruits it to these cortical sites via a conserved region in its C-terminus. I found that Fmp27/Ypr117w and Ybl086c are not required for the growth of cells in response to various stresses such as temperature, salt, osmolarity and detergents. Using fluorescent lipid probes, I found that these proteins do not have a clear role in the distribution of the glycerophospholipids phosphatidylserine (PS), phosphatidylinositol-4-phosphate (PI4P) and phosphatidylinositol-4,5-bisphosphate PI(4,5)P₂ in cells. Instead, I found that the deletion of Fmp27/Ypr117w and its binding partner confer sensitivity to the drug amphotericin B, indicating elevated levels of accessible sterols at the plasma membrane. This indicates a role for these proteins in lipid homeostasis.Medicine, Faculty ofGraduat
Uncovering factors implicated in oncogenic NRAS modification by analyzing large scale functional genomic data sets
No full textThe RAS proteins are a family of small GTPases responsible for cellular signal transduction. Oncogenic mutations in RAS family members (KRAS, HRAS, NRAS) are associated with specific cancers: for example, NRAS is a common driver in melanoma and leukemia. NRAS is trafficked by the addition of a 16-carbon lipid palmitate. The addition and removal of this palmitate are essential for localization and function, and blocking depalmitoylation reduces the proliferation of acute myeloid leukemia blasts in mice carrying oncogenic NRAS. The three isoforms of the alpha/beta hydrolase domain-containing 17 proteins, ABHD17A, ABHD17B and ABHD17C, can remove palmitate from NRAS when expressed in cultured cells. However, it is not known which ABHD17 isoform is most important for oncogenic NRAS activity or if these depalmitoylase enzymes work redundantly. To determine the relative importance of known depalmitoylation and palmitoylation enzymes for NRAS signaling, we used publicly available data from CRISPR-Cas9 functional genomic screens of cancer cell lines to predict genes essential for oncogenic NRAS-driven proliferation. In these screens, each gene is given a gene effect score that represents its essentiality in every cell line. Because KRAS and HRAS compensate for the loss of wild type NRAS, NRAS knockout specifically affects the subset of cell lines whose growth is driven by oncogenic NRAS. Thus, a gene essential for NRAS activity is expected to have a positively correlated gene essentiality score with that of NRAS in these cell lines. We further predict that enzymes required for NRAS palmitoylation and depalmitoylation will be important for the growth of cell lines dependent on oncogenic NRAS. To test these predictions, we separated cell lines into subsets based on the presence or absence of oncogenic NRAS alleles. We then computed Pearson correlation scores for all known genes with respect to NRAS in each subset. As predicted, NRAS lines were enriched for known components of the NRAS signalling pathway. We found that ABHD17B was a top hit in this analysis, and of all known depalmitoylase enzymes, only ABHD17B and ABHD13 had a gene essentiality score that was significantly positively correlated with that of oncogenic NRAS.Medicine, Faculty ofMedical Genetics, Department ofGraduat
Mechanisms that regulate the activity of the NRas depalmitoylase ABHD17
No full textS-acylation is the reversible addition of a lipid to a protein, typically the 16-carbon lipid palmitate. The dynamic nature of palmitoylation enables cycling of signaling molecules, such as NRas, to maintain the plasma membrane localization that is required for oncogenic growth. The alpha/beta hydrolase domain-containing (ABHD) 17 proteins remove palmitate from protein substrates, including NRas, and thus present a promising anti-cancer therapeutic target. The ABHD17 proteins localize to the plasma membrane through a palmitoylated N-terminus, which is required for enzyme activity, but the other mechanisms that regulate activity are currently unknown. Understanding the regulation of the ABHD17 proteins will be crucial to elucidate their role in cell function and disease.
Here, I used in vitro cell-based assays combined with molecular dynamics (MD) simulations performed in collaboration with Dr. S. Vanni and J. Sapia (University of Fribourg) to uncover the regulatory mechanisms of ABHD17. We have shown that the N-terminus of ABHD17A creates a predicted helix that forms the minimal region required for palmitoylation. This palmitoylated N-terminal helix is necessary and sufficient for plasma membrane localization, yet replacement with a plasma membrane anchor does not restore activity, suggesting the N-terminus has an additional function. Replacing the N-terminal palmitoylated cysteines with hydrophobic residues restores activity to ABHD17A when paired with a plasma membrane anchor, suggesting that hydrophobic interactions of this helix with the plasma membrane are critical for activity.
Predictive structural analysis was used to discover a loop structure near the putative lipid-binding pocket of ABHD17. Systematic mutational analysis of this region identified three bulky hydrophobic residues that were required for activity. Importantly, mutations in either the N-terminal helix or the loop structure altered membrane association in MD simulations, and binding pocket conformation, as measured through inhibitor binding assays. This supports the model that the palmitoylated N-terminus recruits and orients ABHD17A at the plasma membrane, allowing the loop to insert in a way that favors substrate binding in the binding pocket. Collectively, these studies identify novel regulatory mechanisms of ABHD17A, which may aid in the creation of anti-cancer therapeutics and contribute to our understanding of related, uncharacterized enzymes.Medicine, Faculty ofMedical Genetics, Department ofGraduat
Membrane contact site targeting and functions of Vps13 and related bridging lipid transporters
No full textSpecialized proteins facilitate inter-organelle lipid transfer at membrane contact sites (MCSs), regions where organelles are tethered in close proximity. The VPS13 proteins constitute a unique class of lipid transporters that simultaneously bridge organelles at MCSs and channel phospholipids between them. Of importance, mutations in each of the four human VPS13 (VPS13A-D) genes are associated with distinct neurological disorders, and it is thought that loss of lipid transport at specific MCSs underlies the cause of these diverse diseases. Understanding how VPS13 proteins target membranes and how their MCS recruitment is regulated is therefore crucial for developing a better understanding of the underlying disease pathologies.
Here, we uncover a conserved VPS13 membrane targeting mechanism using the Saccharomyces cerevisiae model that has a single Vps13 which localizes to multiple organelles and contact sites. We identify a shared motif in the three known organelle-specific adaptors that recruit Vps13, and establish the necessity of these motifs for interaction. We find all adaptors interact at an uncharacterized six-repeat domain that we named the Vps13 Adaptor Binding (VAB) domain. We demonstrate VAB domain repeats 5–6 are sufficient for adaptor interaction and recruitment supporting a model where adaptors compete for binding to a single pocket. Importantly, modeling a spastic ataxia VPS13D missense mutation in this pocket blocks adaptor interactions, Vps13 recruitment, and further results in a vacuolar protein secretion defect that is not associated with any of the known adaptors. These findings support a conserved adaptor binding role for the VAB domain and suggest the presence of as-yet unidentified adaptors in both yeast and humans.
Our studies further identify Fmp27/Hob1 and Ypr117w/Hob2 as putative Vps13 structural homologs localized at endoplasmic reticulum (ER)-plasma membrane (PM) contacts. Recruitment of ER-anchored Fmp27 to PM contacts similarly relies on interactions with a previously uncharacterized protein. Importantly, loss of this PM interactor or of both putative paralogous lipid transporters results in a redistribution of accessible sterols supporting a shared role in lipid homeostasis. Collectively, these findings support the classification of Fmp27 and Hob2 as Vps13-like lipid transporters and suggest adaptor-mediated recruitment is a common membrane targeting strategy for this class of proteins.Medicine, Faculty ofMedical Genetics, Department ofGraduat
Vesicle Transport: Springing the TRAPP
No full textSummaryWhen a coated transport vesicle docks with its target membrane, the coat proteins and docking machinery must be released before the membranes can fuse. A recent paper shows how this disassembly is triggered at precisely the right time
The Vps13 Family of Lipid Transporters and Its Role at Membrane Contact Sites
No full textThe 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.Medicine, Faculty ofMedical Genetics, Department ofReviewedFacultyResearche
