Doctor of Philosophy in Medical Genetics (PhD)
Identifying novel adaptors and localization mechanisms of the neurological disease-associated, Vps13
The goal of my research is to identify the regulatory proteins that control vesicle transport in the yeast Saccharomyces cerevisiae, in order to understand how underlying defects in protein and lipid trafficking contribute to human disease. Vesicle transport is required to switch off signaling receptors that would otherwise promote unregulated cell growth. Retrograde transport also carries signals that promote the continued survival of neurons, and defects in the machinery responsible for retrograde transport are believed to be a cause of motor neuron disease. Because vesicle transport processes are highly conserved, they can be studied in a very simple organism, and the findings applied directly to the study of human cells. Yeast genetics is therefore a powerful tool for the discovery of fundamental cellular mechanisms relevant to human health.
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
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.
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.
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.
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.
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.
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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Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
The 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.
Polytopic membrane protein synthesis involves translation followed by translocation across the lipid bilayer at the ER. Here, various chaperones, together with enzymes that add post and co-translational modifications, help the protein achieve a final three-dimensional structure. General and substrate-specific chaperones prevent toxic aggregation of proteins by shielding and preventing interaction between non-native species. Terminally misfolded proteins are destroyed by the quality control machinery of the cell and the amino acids are recycled for further use. In the following study, we used Chitin synthase III (Chs3) of Saccharomyces cerevisiae as a model to dissect the complexities involved in polytopic membrane protein synthesis at the ER. Previous genetic screens from our lab have revealed a novel regulator of Chs3 trafficking called Pfa4, a DHHC enzyme required for Chs3 palmitoylation at the ER. At the ER, Chs3 also requires Chs7, a dedicated chaperone for folding and assembly. We identified a novel secondary role for Chs7 in Chs3 trafficking as a co-factor required for Chs3 function at the plasma membrane. Our study also examined the role of palmitoylation in Chs3 trafficking. Palmitoylation of Chs3 is required for its efficient interaction with Chs7, in addition to folding and ER export. A genome-wide screen also identified the Ubp3/Bre5 deubiquitination complex as a regulator of non-lipidated Chs3 degradation at the ER. The discovery that dedicated chaperones can take on additional roles and that palmitoylation can influence chaperone-client interactions could provide insights into the workings of the protein folding machinery at the ER.
Rho GTPases are conserved signaling molecules that regulate a wide range of cellular pathways. Numerous regulators and effectors of Rho contribute to the complexity of Rho signaling in cells. Changes in Rho mediated pathways can often lead to human diseases, including cancer and neurodegenerative disorders. How Rho signaling specificity is regulated is not well understood. Our study uses the model organism Saccharomyces cerevisiae to study the regulation of Rho1 signaling. Rho1, the homolog of mammalian RhoA, is a monomeric Rho GTPase that regulates multiple pathways to collectively contribute to cell wall biogenesis. More than fifteen upstream regulators and downstream effectors have been characterized to mediate Rho1 signaling. A genome wide screen was previously conducted in our lab to identify novel regulators of chitin synthase 3 (Chs3) trafficking by measuring the level of chitin at the cell surface. Rho1 signaling has been implicated in the expression and post translational trafficking of Chs3 via the cell wall integrity (CWI) pathway. Not surprisingly, the top hits from the screen included known regulators of the CWI pathway. The screen also uncovered a new component of the CWI pathway, the putative ORF ADC2. Adc2 was physically associated with the RhoGEF Tus1, but not Rom2. It contributed to the localization of Tus1 at the bud neck. Adc2 was also functionally associated with Tus1 in regulating Rho1 signaling. The function of Tus1, but not Rom2, appeared to be dependent on Adc2. Overall, this study identified Adc2 as a novel regulator of Rho1 signaling. Understanding its specific affinity for Tus1 but not Rom2 may offer insights into the signaling specificity of Rho1. The discovery of Adc2 also raises awareness that additional accessory proteins may be associated with Rho signaling not only in yeast but in humans as well.
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