Elizabeth Conibear

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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S-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.

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The 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.

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Eukaryotic 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.

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Specialized 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.

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Clathrin-coated vesicles (CCVs) traffic many cargo proteins throughout the cell to their functional locations. At the center of Golgi/endosome CCV transport is the heterotetrameric AP-1 adaptor protein complex, which coordinates cargo selection and vesicle formation. AP-1 is regulated by a suite of accessory proteins but their identity and functions are incompletely characterized. Here, we identified new AP-1 regulators through targeted yeast genome-wide screens performed in the Conibear lab. Adaptor protein complexes can contain variant subunits but the effect of this subunit exchange is unclear. In yeast, the functional relationship between the “classical” AP-1 complex containing the medium cargo-selective subunit Apm1 and the variant AP-1 complex (AP-1R) containing Apm2 is unclear. Our genome-wide screens indicated that they sort different cargo, and we found they also respond differently to small molecule inhibitors. We identified Mil1 as a novel specific regulator of the Apm2-containing complex, with active-site mutants supporting its role as a lipase. The data are consistent with a model where AP-1 and AP-1R are recruited to distinct membrane areas, facilitating different trafficking pathways.The second screen, for components involved in the trafficking of the AP-1 cargo Chs3, revealed a connection between the previously-identified AP-1 regulator Laa1 and an uncharacterized ORF that we named Laa2. Laa2 bridges AP-1 and Laa1 through an FGxF gamma-adaptin ear binding motif. Our identification of a yeast “Laa complex” consisting of Laa1, Laa2 and the short coiled-coil protein Slo1 led to the discovery of a similar complex in mammalian cells, consisting of HEATR5A, fasciculation and elongation protein zeta 2 (Fez2) and SCOC. We further showed that HEATR5A is distinct from HEATR5B, which works in the aftiphilin/γ-synergin complex previously implicated in AP-1 function. We found a conserved binding site in Laa2, Fez2, aftiphilin and the aftiphilin-related protein CLBA1 for HEATR5-family proteins, providing a new link between various trafficking pathways.This study identifies novel regulatory proteins that may facilitate AP-1 recruitment and function in particular pathways, and illustrates that proteins are not often purely redundant. Apm1 and Apm2 likely sort different cargo as part of distinct AP-1 isoforms, and HEATR5A and HEATR5B participate in distinct complexes.

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Cellular protein trafficking, the concerted action of moving proteins to the appropriate cellular location is important for the proper functioning of the cell. Proteins are sorted at hubs such as the endosome, where they are targeted either for degradation or for recycling to the Golgi. Yeast is an excellent model organism to study protein trafficking due to the conservation with higher eukaryotes and the ease of genetic manipulation. In this thesis, two different high-throughput approaches were used to study the protein machinery that controls the yeast endosomal sorting network.First, correlation analysis, which compares genome-wide phenotypic profiles, was used to uncover new aspects of endosomal sorting and two specific examples were chosen for further study. In the first example, we explored positive and negative genetic interaction profile correlations for members of the uncharacterized yeast complex, BLOC, which suggested a role in endosomal sorting. In particular, we found that BLOC was needed for proper downregulation of a manganese transporter similar to ESCRT (Endosomal Sorting Complex Required for Transport, a well-studied endosomal sorting complex. In the second example, we explored the predictive value of negatively correlated genetic interaction profiles for gene mutants within known protein complexes and found that these negative correlations described two types of regulatory interactions between the resulting proteins, direct inhibition and competition for shared subunits. We then showed that the previously uncharacterized VID Associated Factor 1 (Vaf1) was negatively correlated with respect to its genetic interactions and downregulated by the VID complex. In summary, correlation analysis provides a robust tool to identify the functional relationship between proteins.Second, a quantitative genome-wide endosomal sorting screen followed by a secondary high-throughput microscopy screen, uncovered novel endosomal regulators of the flippase, Neo1. Loss of these regulators caused three phenotypic outcomes: reduced recycling from endosomes, delayed endosomal progression, or reduced colocalization with known binding partners. In particular, a short motif in the N-terminus of Neo1 was found to be necessary for Snx3 dependent sorting, correct sorting of other Snx3 cargos, and full Neo1 function. Overall, this study illuminates the power of high-throughput screens to discover new regulators of endosomal sorting.

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Protein palmitoylation represents the only reversible lipid modification in the cell. As a post-translational modification, it is highly dynamic and plays an important role in protein trafficking and localization. Two families of enzymes mediate dynamic palmitoylation: palmitoyl-acyl transferases (PATs) catalyze palmitate addition, and acyl-protein thioesterases (APTs) catalyze palmitate removal. In mammalian cells, twenty-three PATs have been identified; however, the mechanisms that regulate their enzymatic activity are largely unexplored. Only two APTs, APT1 and APT2, have been identified, but it is unclear if these enzymes act constitutively on all palmitoylated proteins, or if additional depalmitoylases exist. To determine if APT1 and APT2 are responsible for the depalmitoylation of all cytosolic substrates, in this dissertation, I first examined the roles of APT1 and APT2 in protein depalmitoylation. Using a dual pulse-chase strategy to compare protein and palmitate half-lives, I found that simultaneous knockdown or inhibition of APT1 and APT2 strongly blocked palmitate removal from the N-terminal domain of Huntingtin (N-HTT), but had no effect on the depalmitoylation of post-synaptic density-95 (PSD-95). By activity-based protein profiling (ABPP), I showed that the APT1/2 inhibitor Palmostatin B has additional serine hydrolase targets that may play a role in PSD-95 depalmitoylation. Moreover, Palmostatin B induced PSD-95-GFP re-distribution in COS-7 cells, a phenotype not observed with APT1- and APT2-selective inhibitors. These results demonstrate that serine hydrolases other than APT1 and APT2 mediate the substrate-specific removal of palmitate from cytosolic proteins. I also investigated a possible novel regulator of the PAT HTT-interacting protein 14 (HIP14), Optineurin (OPTN), a cargo adaptor known to interact with HTT to mediate post-Golgi vesicle trafficking. I validated this interaction by co-immunoprecipitation and showed that OPTN is not a palmitoylated substrate. Furthermore, HIP14, OPTN, and HTT formed a trimeric complex. I mapped the binding of OPTN and HTT to the HIP14 ankyrin repeat domain, and identified mutations that selectively destabilized the HIP14/OPTN interaction. I hypothesize that OPTN transports HIP14 to distinct subcellular compartments to regulate its access to substrates. In summary, these results reveal potential novel regulatory components in the dynamic palmitoylation cycle.

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In Eukaryotes, luminal and transmembrane proteins are moved to their functional locations by conserved membrane trafficking machinery. In this process, cargo adaptors bind motifs present on cargo, indirectly linking the proteins to coats, which deform membranes and form transport vesicles. Here, cargo adaptor recruitment and cargo recognition was studied by characterizing associated factors in the budding yeast Saccharomyces cerevisiae. Possible cargo adaptor-associated factors were identified in a proteomics study that grouped protein-protein interactions into 501 putative membrane associated complexes using a Markov clustering algorithm. Two clusters were selected for this work.The first contained the uncharacterized protein Ssp120 with the endoplasmic reticulum-to-Golgi trafficking complex Emp46/Emp47. Ssp120 stably interacted with the Emp46/Emp47 complex and depended on Emp47 for its punctate localization. The C-terminus of Ssp120 mediated the interaction. Homology with human MCFD2 suggests that Ssp120 may link a subset of cargo to Emp46/Emp47.The second cluster was comprised of retromer, an endosome-to-Golgi trafficking complex, and the Rab5-family guanine nucleotide exchange factor (GEF) Muk1. Both Muk1 and the other known Rab5-family GEF, Vps9, interacted with retromer and the presence of at least one was required for retromer recruitment to endosomes. Additionally, a new VPS9 domain-containing protein present was identified and shown to complement loss of MUK1 and VPS9. Retromer recruitment was shown to be dependent on putative GEF catalytic residues and the presence of their target Rabs. Furthermore, loss of GEFs resulted in mislocalization of the potential Rab5-family GTPase effector, Vps34, and its lipid product, phosphatidylinositol 3-phosphate (PI3P), to the vacuolar membrane. As retromer is recruited by PI3P, the data support a positive feedback model whereby retromer interacts with GEFs to indirectly modify the lipid composition of the membrane allowing further localized recruitment.This study validates the approach of studying novel interactors of cargo recognition complexes to better understand their function. It suggests that Ssp120 may recognize a subset of Emp46-Emp47 cargo, indicating that an associated factor can diversify the proteins recognized by a given cargo adaptor. Furthermore, the work on retromer suggests a novel mechanism for the reinforcement of cargo selective complex recruitment that may be conserved in humans.

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The process of endocytic recycling, in which cell surface proteins are internalized and re-delivered to the plasma membrane, is essential in all eukaryotes for maintaining plasma membrane composition and regulating the surface levels of signaling receptors. The applicability of Saccharomyces cerevisiae as a model to study endocytic recycling is a subject of debate, as there appears to be critical differences between yeast and mammalian cells. For example, while clathrin and its adaptors are critical for uptake in mammals, they do not seem to be essential in yeast. Endocytic recycling has not been comprehensively studied on a genetic level in yeast, and only limited cargo have been considered, making it difficult to accurately assess the similarity between the two systems. Furthermore, the transport of SNARE proteins is poorly understood, but appears to involve specialized mechanisms. This study uses a genome-wide screening approach to systematically and quantitatively identify genes required for the endocytic recycling of the yeast SNARE protein Snc1, homologous to the mammalian VAMP2/synaptobrevin.Endocytic defects for mutants of many yeast homologs of mammalian endocytosis genes were identified, for the first time. Significantly, a cargo-selective and partially-redundant role for clathrin and its adaptors yAP1801 and yAP1802 was identified. The lipid phosphatase Inp52 was found to mediate AP180 release from endocytic vesicles. Additionally, the previously uncharacterized protein Ldb17, homologous to the mammalian endocytic protein SPIN90, was identified as a new component of the endocytic machinery, and regulates both coat and actin dynamics at endocytic sites.Factors regulating Snc1 recycling were also identified, including the variant clathrin adaptor AP-1R. This is the first reported function for this complex. The previously uncharacterized protein Ima1 was found to be a putative enzyme that specifically binds to AP-1R, and may have activity related to AP-1R function.Overall, this study demonstrates that endocytic recycling in yeast and mammals is more similar than previously appreciated, and identifies new factors in this process. Furthermore, it raises awareness of the degree of cargo-selectivity underlying this pathway, and demonstrates quantitative methods that can be further applied to future studies in both systems.

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Proteins and lipids are selectively transported between the Golgi, plasma membrane and endosomes by a network of vesicle-mediated endosomal transport pathways. Trafficking specificity requires the coordination of multiple protein assemblies and signals of compartment identity. Genetic screens, and molecular and biochemical techniques, have revealed many components for endosomal transport, but questions regarding the mechanisms of specificity and the coordination of trafficking pathways remain. The Golgi Associated Retrograde Protein (GARP) complex is required to tether vesicles derived from multiple types of endosomes with the Golgi. In the absence of GARP, retrograde transport from endosomes to the Golgi is abolished, and numerous cargoes are missorted. Mutation of the GARP subunit Vps54 causes motor neuron disease in the mouse, emphasizing the physiological importance of GARP. Tethering requires recognition of multiple membranes, but how GARP recognizes vesicles derived from multiple upstream compartments is not known. In my first body of work, the function of the GARP subunit Vps54 was addressed. The N-terminal portion of Vps54 was found to be important for GARP complex assembly and stability, while the C-terminal portion localized to a compartment with features of an early endosome. In the absence of the C-terminal domain, retrieval of early endosome cargo became dependent on late-endosome retrograde transport. This body of work supports the model that tethers recognize, and possibly distinguish between, upstream compartments. The machinery involved in retrograde transport from endosomes is not fully understood. In my second body of work, genes involved endosomal transport were systematically identified by screening mutant collections with a reporter of early endosome dysfunction. To evaluate the relationships between genes and pathways discovered in this screen, genetic interaction analyses with two phenotypes, growth and endosomal dysfunction, were performed. An analysis of genetic interactions based on trafficking dysfunction revealed interesting genetic relationships between endosomal coat proteins and their regulators. This body of work provides insight into the relationships between endosomal transport pathways and presents a framework to discover relationships between genes and pathways discovered in a genomic screen. Together, this thesis presents a molecular and pathway perspective of endosomal transport that provides insight into pathway specificity and the relationships between pathway components.

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No abstract available.