{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/109555"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/109555","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"The regulation of yeast homotypic vacuole fusion and membrane trafficking events by sphingolipids, ergosterol, and lipid rafts","abstract":"Membrane fusion is a conserved process that facilitates essential events like endocytosis, secretion, cell division and autophagy. Membrane fusion occurs at all organelles, and many of the fundamental processes required for fusion have been extensively studied. This process requires a minimal set of proteins and lipids in fully reconstituted systems, but in vivo fusion is a highly regulated process, with inputs from multiple signaling pathways. In live cells, membrane fusion requires the activity of SNAREs, small GTPases, chaperones, tethering complexes, and specific regulatory lipids. This process occurs in four experimentally defined steps: priming, tethering, docking, and fusion. Work with baker’s yeast identified interdependence between a set of pro-fusion proteins and regulatory lipids at the vacuole, wherein both interacting partners are required for proper localization to specific membrane domains. Regulatory lipids and select fusion proteins enrich in the vertex ring domain of vacuoles that are in the process of tethering and docking together, which can be visualized with microscopic techniques. While there has been significant progress made in the studies of glycerophospholipids as regulatory lipids, there are few such studies focused on sphingolipids. In the work below, we present initial studies that show that sphingolipids specifically regulate the tethering and docking steps of homotypic vacuole fusion, they are necessary for proper AP-3 trafficking, and they influence cell division processes. The Saccharomyces cerevisiae lysosome-like vacuole is a useful model for studying membrane fusion events and organelle maturation processes utilized by all eukaryotes. The vacuolar membrane is capable of forming micrometer and nanometer scale domains that can be visualized using microscopic techniques and segregate into regions with surprisingly distinct lipid and protein compositions. These lipid raft domains are liquid-ordered (Lo) like regions that are rich in sphingolipids, phospholipids with saturated acyl chains, and ergosterol. Recent studies have shown that these lipid rafts contain an enrichment of many different proteins that function in essential activities such as nutrient transport, organelle contact, membrane trafficking, and homotypic fusion, suggesting that they are biologically relevant regions within the vacuole membrane. Here, we discuss recent developments and the current understanding of sphingolipid and ergosterol function at the vacuole, the composition and function of lipid rafts at this organelle and how the distinct lipid and protein composition of these regions facilitates the biological processes outlined above. Sphingolipids are essential factors in membrane trafficking and eukaryotic cellular homeostasis. This is exemplified by numerous mammalian lysosomal disorders that are linked to defects in complex sphingolipid metabolic pathways. Here, we present evidence that sphingolipids containing very long-chain fatty acids (VLCFAs) function as positive regulators of homotypic vacuolar fusion. Yeast lacking the C26 VLCFA elongase Elo3p produce irregular vacuoles that are unable to fuse at wild type levels. We found that elo3Δ yeast mislocalize a number of fusion regulators including the Rab Ypt7p, which is depleted from the vertex domains of docked vacuoles where fusion occurs. In addition, we show that SNARE complex formation was attenuated on elo3Δ vacuoles. While protein localization was strongly affected in the absence of Elo3p, the vertex localization of other lipids, including ergosterol, was only moderately affected. Yet, we found that the fluidity of elo3Δ vacuoles was significantly increased. Together these data suggest that C26 VLCFA sphingolipids act as regulators of homotypic vacuolar fusion, likely by assembling membrane microdomains that promote the enrichment of protein machinery required tethering and docking.","abstract_html":"Membrane fusion is a conserved process that facilitates essential events like endocytosis, secretion, cell division and autophagy. Membrane fusion occurs at all organelles, and many of the fundamental processes required for fusion have been extensively studied. This process requires a minimal set of proteins and lipids in fully reconstituted systems, but in vivo fusion is a highly regulated process, with inputs from multiple signaling pathways. In live cells, membrane fusion requires the activity of SNAREs, small GTPases, chaperones, tethering complexes, and specific regulatory lipids. This process occurs in four experimentally defined steps: priming, tethering, docking, and fusion. Work with baker’s yeast identified interdependence between a set of pro-fusion proteins and regulatory lipids at the vacuole, wherein both interacting partners are required for proper localization to specific membrane domains. Regulatory lipids and select fusion proteins enrich in the vertex ring domain of vacuoles that are in the process of tethering and docking together, which can be visualized with microscopic techniques. While there has been significant progress made in the studies of glycerophospholipids as regulatory lipids, there are few such studies focused on sphingolipids. In the work below, we present initial studies that show that sphingolipids specifically regulate the tethering and docking steps of homotypic vacuole fusion, they are necessary for proper AP-3 trafficking, and they influence cell division processes. The Saccharomyces cerevisiae lysosome-like vacuole is a useful model for studying membrane fusion events and organelle maturation processes utilized by all eukaryotes. The vacuolar membrane is capable of forming micrometer and nanometer scale domains that can be visualized using microscopic techniques and segregate into regions with surprisingly distinct lipid and protein compositions. These lipid raft domains are liquid-ordered (Lo) like regions that are rich in sphingolipids, phospholipids with saturated acyl chains, and ergosterol. Recent studies have shown that these lipid rafts contain an enrichment of many different proteins that function in essential activities such as nutrient transport, organelle contact, membrane trafficking, and homotypic fusion, suggesting that they are biologically relevant regions within the vacuole membrane. Here, we discuss recent developments and the current understanding of sphingolipid and ergosterol function at the vacuole, the composition and function of lipid rafts at this organelle and how the distinct lipid and protein composition of these regions facilitates the biological processes outlined above. Sphingolipids are essential factors in membrane trafficking and eukaryotic cellular homeostasis. This is exemplified by numerous mammalian lysosomal disorders that are linked to defects in complex sphingolipid metabolic pathways. Here, we present evidence that sphingolipids containing very long-chain fatty acids (VLCFAs) function as positive regulators of homotypic vacuolar fusion. Yeast lacking the C26 VLCFA elongase Elo3p produce irregular vacuoles that are unable to fuse at wild type levels. We found that elo3Δ yeast mislocalize a number of fusion regulators including the Rab Ypt7p, which is depleted from the vertex domains of docked vacuoles where fusion occurs. In addition, we show that SNARE complex formation was attenuated on elo3Δ vacuoles. While protein localization was strongly affected in the absence of Elo3p, the vertex localization of other lipids, including ergosterol, was only moderately affected. Yet, we found that the fluidity of elo3Δ vacuoles was significantly increased. Together these data suggest that C26 VLCFA sphingolipids act as regulators of homotypic vacuolar fusion, likely by assembling membrane microdomains that promote the enrichment of protein machinery required tethering and docking.","abstract_has_math":false,"creators":["Hurst, Logan Robert"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Fratti, Rutilio A","Gennis, Robert B","Procko, Erik","Zhang, Kai"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021-03-05T21:45:07Z","date_published":"2021-03-05T21:45:07Z","updated_at":"2026-07-22T22:24:50Z","subjects":["membrane fusion, sphingolipids, lipid rafts, Rab GTPase, trafficking"],"languages":["en"],"rights":["Copyright 2020 Logan Hurst"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/109555","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Fratti, Rutilio A","Gennis, Robert B","Procko, Erik","Zhang, Kai"]},{"key":"dc:creator","label":"Author","values":["Hurst, Logan Robert"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2021-03-05T21:45:07Z","2023-03-05T21:47:41Z","2020-08-27","2020-12"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["membrane fusion, sphingolipids, lipid rafts, Rab GTPase, trafficking"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2020 Logan Hurst"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/109555"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Membrane fusion is a conserved process that facilitates essential events like endocytosis, secretion, cell division and autophagy. Membrane fusion occurs at all organelles, and many of the fundamental processes required for fusion have been extensively studied. This process requires a minimal set of proteins and lipids in fully reconstituted systems, but in vivo fusion is a highly regulated process, with inputs from multiple signaling pathways. In live cells, membrane fusion requires the activity of SNAREs, small GTPases, chaperones, tethering complexes, and specific regulatory lipids. This process occurs in four experimentally defined steps: priming, tethering, docking, and fusion. Work with baker’s yeast identified interdependence between a set of pro-fusion proteins and regulatory lipids at the vacuole, wherein both interacting partners are required for proper localization to specific membrane domains. Regulatory lipids and select fusion proteins enrich in the vertex ring domain of vacuoles that are in the process of tethering and docking together, which can be visualized with microscopic techniques. While there has been significant progress made in the studies of glycerophospholipids as regulatory lipids, there are few such studies focused on sphingolipids. In the work below, we present initial studies that show that sphingolipids specifically regulate the tethering and docking steps of homotypic vacuole fusion, they are necessary for proper AP-3 trafficking, and they influence cell division processes. The Saccharomyces cerevisiae lysosome-like vacuole is a useful model for studying membrane fusion events and organelle maturation processes utilized by all eukaryotes. The vacuolar membrane is capable of forming micrometer and nanometer scale domains that can be visualized using microscopic techniques and segregate into regions with surprisingly distinct lipid and protein compositions. These lipid raft domains are liquid-ordered (Lo) like regions that are rich in sphingolipids, phospholipids with saturated acyl chains, and ergosterol. Recent studies have shown that these lipid rafts contain an enrichment of many different proteins that function in essential activities such as nutrient transport, organelle contact, membrane trafficking, and homotypic fusion, suggesting that they are biologically relevant regions within the vacuole membrane. Here, we discuss recent developments and the current understanding of sphingolipid and ergosterol function at the vacuole, the composition and function of lipid rafts at this organelle and how the distinct lipid and protein composition of these regions facilitates the biological processes outlined above. Sphingolipids are essential factors in membrane trafficking and eukaryotic cellular homeostasis. This is exemplified by numerous mammalian lysosomal disorders that are linked to defects in complex sphingolipid metabolic pathways. Here, we present evidence that sphingolipids containing very long-chain fatty acids (VLCFAs) function as positive regulators of homotypic vacuolar fusion. Yeast lacking the C26 VLCFA elongase Elo3p produce irregular vacuoles that are unable to fuse at wild type levels. We found that elo3Δ yeast mislocalize a number of fusion regulators including the Rab Ypt7p, which is depleted from the vertex domains of docked vacuoles where fusion occurs. In addition, we show that SNARE complex formation was attenuated on elo3Δ vacuoles. While protein localization was strongly affected in the absence of Elo3p, the vertex localization of other lipids, including ergosterol, was only moderately affected. Yet, we found that the fluidity of elo3Δ vacuoles was significantly increased. Together these data suggest that C26 VLCFA sphingolipids act as regulators of homotypic vacuolar fusion, likely by assembling membrane microdomains that promote the enrichment of protein machinery required tethering and docking.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2022-12-01","The student, Logan Hurst, accepted the attached license on 2020-08-24 at 14:00.","The student, Logan Hurst, submitted this Dissertation for approval on 2020-08-24 at 14:11.","This Dissertation was approved for publication on 2020-08-27 at 11:06.","DSpace SAF Submission Ingestion Package generated from Vireo submission #15779 on 2021-03-04 at 16:30:00","Made available in DSpace on 2021-03-05T21:45:07Z (GMT). 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Membrane fusion occurs at all organelles, and many of the fundamental processes required for fusion have been extensively studied. This process requires a minimal set of proteins and lipids in fully reconstituted systems, but in vivo fusion is a highly regulated process, with inputs from multiple signaling pathways. In live cells, membrane fusion requires the activity of SNAREs, small GTPases, chaperones, tethering complexes, and specific regulatory lipids. This process occurs in four experimentally defined steps: priming, tethering, docking, and fusion. Work with baker’s yeast identified interdependence between a set of pro-fusion proteins and regulatory lipids at the vacuole, wherein both interacting partners are required for proper localization to specific membrane domains. Regulatory lipids and select fusion proteins enrich in the vertex ring domain of vacuoles that are in the process of tethering and docking together, which can be visualized with microscopic techniques. While there has been significant progress made in the studies of glycerophospholipids as regulatory lipids, there are few such studies focused on sphingolipids. In the work below, we present initial studies that show that sphingolipids specifically regulate the tethering and docking steps of homotypic vacuole fusion, they are necessary for proper AP-3 trafficking, and they influence cell division processes. The Saccharomyces cerevisiae lysosome-like vacuole is a useful model for studying membrane fusion events and organelle maturation processes utilized by all eukaryotes. The vacuolar membrane is capable of forming micrometer and nanometer scale domains that can be visualized using microscopic techniques and segregate into regions with surprisingly distinct lipid and protein compositions. These lipid raft domains are liquid-ordered (Lo) like regions that are rich in sphingolipids, phospholipids with saturated acyl chains, and ergosterol. Recent studies have shown that these lipid rafts contain an enrichment of many different proteins that function in essential activities such as nutrient transport, organelle contact, membrane trafficking, and homotypic fusion, suggesting that they are biologically relevant regions within the vacuole membrane. Here, we discuss recent developments and the current understanding of sphingolipid and ergosterol function at the vacuole, the composition and function of lipid rafts at this organelle and how the distinct lipid and protein composition of these regions facilitates the biological processes outlined above. Sphingolipids are essential factors in membrane trafficking and eukaryotic cellular homeostasis. This is exemplified by numerous mammalian lysosomal disorders that are linked to defects in complex sphingolipid metabolic pathways. Here, we present evidence that sphingolipids containing very long-chain fatty acids (VLCFAs) function as positive regulators of homotypic vacuolar fusion. Yeast lacking the C26 VLCFA elongase Elo3p produce irregular vacuoles that are unable to fuse at wild type levels. We found that elo3Δ yeast mislocalize a number of fusion regulators including the Rab Ypt7p, which is depleted from the vertex domains of docked vacuoles where fusion occurs. In addition, we show that SNARE complex formation was attenuated on elo3Δ vacuoles. While protein localization was strongly affected in the absence of Elo3p, the vertex localization of other lipids, including ergosterol, was only moderately affected. Yet, we found that the fluidity of elo3Δ vacuoles was significantly increased. Together these data suggest that C26 VLCFA sphingolipids act as regulators of homotypic vacuolar fusion, likely by assembling membrane microdomains that promote the enrichment of protein machinery required tethering and docking.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2022-12-01","The student, Logan Hurst, accepted the attached license on 2020-08-24 at 14:00.","The student, Logan Hurst, submitted this Dissertation for approval on 2020-08-24 at 14:11.","This Dissertation was approved for publication on 2020-08-27 at 11:06.","DSpace SAF Submission Ingestion Package generated from Vireo submission #15779 on 2021-03-04 at 16:30:00","Made available in DSpace on 2021-03-05T21:45:07Z (GMT). No. of bitstreams: 2 HURST-DISSERTATION-2020.pdf: 66026081 bytes, checksum: 5c33d561f6f1c39f50efd32f78489618 (MD5) LICENSE.txt: 4208 bytes, checksum: 606fd8be98ffe0d4a21ae227d37bacd0 (MD5) Previous issue date: 2020-08-27","Embargo set by: Seth Robbins for item 117260 Lift date: 2023-03-05T21:45:47Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 117260 Lift date: 2023-03-05T21:47:41Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited"],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/2142/109555"],"dc:language":["en"],"dc:rights":["Copyright 2020 Logan Hurst"],"dc:subject":["membrane fusion, sphingolipids, lipid rafts, Rab GTPase, trafficking"],"dc:title":["The regulation of yeast homotypic vacuole fusion and membrane trafficking events by sphingolipids, ergosterol, and lipid rafts"],"dc:type":["text","Thesis"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:24:50Z"}