{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/390001"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/390001","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Cryo-EM structural exploration of human VPS34 complexes and their activation by RAB GTPases and NRBF2","abstract":"VPS34 is a primordial lipid kinase that catalyses the conversion of lipid phosphatidylinositol (PI) into phosphatidylinositol-3-phosphate (PI3P) on membranes. The reaction product, PI3P, is a potent signalling lipid involved promoting autophagy, phagocytosis, and endocytic sorting in mammalian cells. In addition to its roles in normal homeostasis, VPS34 can be hijacked by viruses and bacteria during infection. VPS34 is a part of two heterotetrameric complexes in cells. Complex I is critical for the initiation step of autophagy, and consists of VPS34, VPS15, Beclin1, and ATG14L. In addition, complex I has a tightly bound fifth subunit NRBF2, although its effect on the primary role of complex I is not clear. Complex II has a role in endocytic sorting and contains the same subunits, except the ATG14L is replaced by UVRAG. VPS34 complexes can be specifically activated by RAB GTPases RAB1A (for complex I) and RAB5A (for complex II), but the mechanistic details of how this specificity is achieved have remained elusive due to a lack of high-resolution structural data for human VPS34 complexes. In this work, I used single particle cryo-EM to determine the structures of VPS34 complexes I and II, both alone and together with their specific RABs. I showed that there are intrinsic differences in the conformation of complexes I and II caused by the complex-specific subunits ATG14L and UVRAG, which result in selective binding of RAB1A by complex I and RAB5A by complex II. Furthermore, the RAB GTPases form specific contacts with VPS34 complexes through their Switch regions. I also discovered that complex II has a second RAB5A binding site on the VPS15 subunit. This site is required for full activation of complex II, implying that complex II can bind two RAB5A molecules simultaneously. In contrast, complex I does not bind a second RAB protein on the VPS15 subunit. This is because in complex I a loop in the Beclin1 subunit is positioned to occlude the VPS15 binding site, whereas in complex II this Beclin1 loop is far away from the RAB binding site. We showed that the RAB binding site on VPS15 is functionally important in yeast, while in mammalian cells the VPS34 site might be the primary site. I showed that NRBF2, a fifth subunit that binds tightly to complex I, activates it under certain conditions. In the absence of RAB1A, neither full length NRBF2, nor its N-terminal MIT domain have a large effect on the complex I kinase activity. By contrast, in the presence of RAB1A, NRBF2 can synergise with the GTPase to greatly boost complex I’s activity. This effect was only observed when the concentration of NRBF2 was lower than complex I. Under the activating conditions, NRBF2 also increases complex I recruitment to giant unilamellar vesicle membranes. We explain this phenomenon by the ability of NRBF2 to dimerise and bridge two complex I + RAB1A assemblies, thereby increasing their affinities for the membrane, which, in turn, boosts the rate of PI3P production. Finally, I used single particle cryo-EM to map the binding sites of eight anti-VPS34 complex nanobodies. The structural information explains the differential functional effects some nanobodies have on complexes I and II. The nanobodies can be used to label VPS34 complexes in in vitro and in vivo experiments, and the structural data can guide the selection of a nanobody most suitable for a particular application. In addition, some nanobodies could be used in cryo-EM to stabilise selected regions of VPS34 complexes and improve local resolution.","abstract_html":"VPS34 is a primordial lipid kinase that catalyses the conversion of lipid phosphatidylinositol (PI) into phosphatidylinositol-3-phosphate (PI3P) on membranes. The reaction product, PI3P, is a potent signalling lipid involved promoting autophagy, phagocytosis, and endocytic sorting in mammalian cells. In addition to its roles in normal homeostasis, VPS34 can be hijacked by viruses and bacteria during infection. VPS34 is a part of two heterotetrameric complexes in cells. Complex I is critical for the initiation step of autophagy, and consists of VPS34, VPS15, Beclin1, and ATG14L. In addition, complex I has a tightly bound fifth subunit NRBF2, although its effect on the primary role of complex I is not clear. Complex II has a role in endocytic sorting and contains the same subunits, except the ATG14L is replaced by UVRAG. VPS34 complexes can be specifically activated by RAB GTPases RAB1A (for complex I) and RAB5A (for complex II), but the mechanistic details of how this specificity is achieved have remained elusive due to a lack of high-resolution structural data for human VPS34 complexes. In this work, I used single particle cryo-EM to determine the structures of VPS34 complexes I and II, both alone and together with their specific RABs. I showed that there are intrinsic differences in the conformation of complexes I and II caused by the complex-specific subunits ATG14L and UVRAG, which result in selective binding of RAB1A by complex I and RAB5A by complex II. Furthermore, the RAB GTPases form specific contacts with VPS34 complexes through their Switch regions. I also discovered that complex II has a second RAB5A binding site on the VPS15 subunit. This site is required for full activation of complex II, implying that complex II can bind two RAB5A molecules simultaneously. In contrast, complex I does not bind a second RAB protein on the VPS15 subunit. This is because in complex I a loop in the Beclin1 subunit is positioned to occlude the VPS15 binding site, whereas in complex II this Beclin1 loop is far away from the RAB binding site. We showed that the RAB binding site on VPS15 is functionally important in yeast, while in mammalian cells the VPS34 site might be the primary site. I showed that NRBF2, a fifth subunit that binds tightly to complex I, activates it under certain conditions. In the absence of RAB1A, neither full length NRBF2, nor its N-terminal MIT domain have a large effect on the complex I kinase activity. By contrast, in the presence of RAB1A, NRBF2 can synergise with the GTPase to greatly boost complex I’s activity. This effect was only observed when the concentration of NRBF2 was lower than complex I. Under the activating conditions, NRBF2 also increases complex I recruitment to giant unilamellar vesicle membranes. We explain this phenomenon by the ability of NRBF2 to dimerise and bridge two complex I + RAB1A assemblies, thereby increasing their affinities for the membrane, which, in turn, boosts the rate of PI3P production. Finally, I used single particle cryo-EM to map the binding sites of eight anti-VPS34 complex nanobodies. The structural information explains the differential functional effects some nanobodies have on complexes I and II. The nanobodies can be used to label VPS34 complexes in in vitro and in vivo experiments, and the structural data can guide the selection of a nanobody most suitable for a particular application. In addition, some nanobodies could be used in cryo-EM to stabilise selected regions of VPS34 complexes and improve local resolution.","abstract_has_math":false,"creators":["Spokaite, Saule"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Williams, Roger"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-01-31","date_published":"2025-01-31","updated_at":"2026-07-22T22:24:24Z","subjects":["VPS34","cryo-EM","RAB1A","NRBF2","RAB5A"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/321e4ddf-a4f5-40cc-a864-ab426ef3741c/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.121704","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Williams, Roger"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["MRC Research Studentship"]},{"key":"dc:creator","label":"Author","values":["Spokaite, Saule"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2025-01-31"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/390001"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["VPS34","cryo-EM","RAB1A","NRBF2","RAB5A"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/321e4ddf-a4f5-40cc-a864-ab426ef3741c/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-09-30"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.121704"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/58a10d8f-a00d-4744-a5da-bb121e58d454/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["VPS34 is a primordial lipid kinase that catalyses the conversion of lipid phosphatidylinositol (PI) into phosphatidylinositol-3-phosphate (PI3P) on membranes. The reaction product, PI3P, is a potent signalling lipid involved promoting autophagy, phagocytosis, and endocytic sorting in mammalian cells. In addition to its roles in normal homeostasis, VPS34 can be hijacked by viruses and bacteria during infection. VPS34 is a part of two heterotetrameric complexes in cells. Complex I is critical for the initiation step of autophagy, and consists of VPS34, VPS15, Beclin1, and ATG14L. In addition, complex I has a tightly bound fifth subunit NRBF2, although its effect on the primary role of complex I is not clear. Complex II has a role in endocytic sorting and contains the same subunits, except the ATG14L is replaced by UVRAG. VPS34 complexes can be specifically activated by RAB GTPases RAB1A (for complex I) and RAB5A (for complex II), but the mechanistic details of how this specificity is achieved have remained elusive due to a lack of high-resolution structural data for human VPS34 complexes. In this work, I used single particle cryo-EM to determine the structures of VPS34 complexes I and II, both alone and together with their specific RABs. I showed that there are intrinsic differences in the conformation of complexes I and II caused by the complex-specific subunits ATG14L and UVRAG, which result in selective binding of RAB1A by complex I and RAB5A by complex II. Furthermore, the RAB GTPases form specific contacts with VPS34 complexes through their Switch regions. I also discovered that complex II has a second RAB5A binding site on the VPS15 subunit. This site is required for full activation of complex II, implying that complex II can bind two RAB5A molecules simultaneously. In contrast, complex I does not bind a second RAB protein on the VPS15 subunit. This is because in complex I a loop in the Beclin1 subunit is positioned to occlude the VPS15 binding site, whereas in complex II this Beclin1 loop is far away from the RAB binding site. We showed that the RAB binding site on VPS15 is functionally important in yeast, while in mammalian cells the VPS34 site might be the primary site. I showed that NRBF2, a fifth subunit that binds tightly to complex I, activates it under certain conditions. In the absence of RAB1A, neither full length NRBF2, nor its N-terminal MIT domain have a large effect on the complex I kinase activity. By contrast, in the presence of RAB1A, NRBF2 can synergise with the GTPase to greatly boost complex I’s activity. This effect was only observed when the concentration of NRBF2 was lower than complex I. Under the activating conditions, NRBF2 also increases complex I recruitment to giant unilamellar vesicle membranes. We explain this phenomenon by the ability of NRBF2 to dimerise and bridge two complex I + RAB1A assemblies, thereby increasing their affinities for the membrane, which, in turn, boosts the rate of PI3P production. Finally, I used single particle cryo-EM to map the binding sites of eight anti-VPS34 complex nanobodies. The structural information explains the differential functional effects some nanobodies have on complexes I and II. The nanobodies can be used to label VPS34 complexes in in vitro and in vivo experiments, and the structural data can guide the selection of a nanobody most suitable for a particular application. 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In addition to its roles in normal homeostasis, VPS34 can be hijacked by viruses and bacteria during infection. VPS34 is a part of two heterotetrameric complexes in cells. Complex I is critical for the initiation step of autophagy, and consists of VPS34, VPS15, Beclin1, and ATG14L. In addition, complex I has a tightly bound fifth subunit NRBF2, although its effect on the primary role of complex I is not clear. Complex II has a role in endocytic sorting and contains the same subunits, except the ATG14L is replaced by UVRAG. VPS34 complexes can be specifically activated by RAB GTPases RAB1A (for complex I) and RAB5A (for complex II), but the mechanistic details of how this specificity is achieved have remained elusive due to a lack of high-resolution structural data for human VPS34 complexes. In this work, I used single particle cryo-EM to determine the structures of VPS34 complexes I and II, both alone and together with their specific RABs. I showed that there are intrinsic differences in the conformation of complexes I and II caused by the complex-specific subunits ATG14L and UVRAG, which result in selective binding of RAB1A by complex I and RAB5A by complex II. Furthermore, the RAB GTPases form specific contacts with VPS34 complexes through their Switch regions. I also discovered that complex II has a second RAB5A binding site on the VPS15 subunit. This site is required for full activation of complex II, implying that complex II can bind two RAB5A molecules simultaneously. In contrast, complex I does not bind a second RAB protein on the VPS15 subunit. This is because in complex I a loop in the Beclin1 subunit is positioned to occlude the VPS15 binding site, whereas in complex II this Beclin1 loop is far away from the RAB binding site. We showed that the RAB binding site on VPS15 is functionally important in yeast, while in mammalian cells the VPS34 site might be the primary site. I showed that NRBF2, a fifth subunit that binds tightly to complex I, activates it under certain conditions. In the absence of RAB1A, neither full length NRBF2, nor its N-terminal MIT domain have a large effect on the complex I kinase activity. By contrast, in the presence of RAB1A, NRBF2 can synergise with the GTPase to greatly boost complex I’s activity. This effect was only observed when the concentration of NRBF2 was lower than complex I. Under the activating conditions, NRBF2 also increases complex I recruitment to giant unilamellar vesicle membranes. We explain this phenomenon by the ability of NRBF2 to dimerise and bridge two complex I + RAB1A assemblies, thereby increasing their affinities for the membrane, which, in turn, boosts the rate of PI3P production. Finally, I used single particle cryo-EM to map the binding sites of eight anti-VPS34 complex nanobodies. The structural information explains the differential functional effects some nanobodies have on complexes I and II. The nanobodies can be used to label VPS34 complexes in in vitro and in vivo experiments, and the structural data can guide the selection of a nanobody most suitable for a particular application. In addition, some nanobodies could be used in cryo-EM to stabilise selected regions of VPS34 complexes and improve local resolution."],"dc:format.checksum.md5":["d3a92ff5d38cffd09dd2223806c7b4da","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.121704"],"dc:identifier.uri":["https://www.repository.cam.ac.uk/bitstreams/58a10d8f-a00d-4744-a5da-bb121e58d454/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/390001"],"dc:rights":["https://www.repository.cam.ac.uk/bitstreams/321e4ddf-a4f5-40cc-a864-ab426ef3741c/download","http://purl.org/NET/rdflicense/allrightsreserved"],"dc:rights.embargodate":["2026-09-30"],"dc:rights.embargotype":["embargo"],"dc:subject":["VPS34","cryo-EM","RAB1A","NRBF2","RAB5A"],"dc:title":["Cryo-EM structural exploration of human VPS34 complexes and their activation by RAB GTPases and NRBF2"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:24Z"}