Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeleton

Detalhes bibliográficos
Autor(a) principal: Braga, Teresa
Data de Publicação: 2021
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10451/53670
Resumo: The spatiotemporal organisation of cells is essential for the correct performance of organisms, which implies a complex and highly regulated system. In eukaryotic cells, this aspect is of particular importance, with the cytoskeleton playing a key role as a determinant of cell architecture and function. Regarding plant cells, the actin cytoskeleton is involved in most developmental processes, regulating membrane trafficking, organelle movement, morphogenesis and the establishment and maintenance of cell polarity. The dynamics of the actin cytoskeleton are regulated by numerous actin-binding proteins (ABPs), that modulate the polymerisation/depolymerisation of filaments, mediate the transport of cellular components, contribute to the formation of higher-order actin structures, and serve as a bridge between the actin cytoskeleton and membrane structures in the cell. The NETWORKED (NET) proteins are a recently characterised, plant-specific superfamily of actin-binding proteins that serve as interfaces between the actin cytoskeleton and various cell membranes. Some members (NET3A, NET3C; NET4A, NET4B) share a conserved domain in the C-terminal region that has at its centre three conserved amino acids – isoleucine (I), arginine (R) and glutamine (Q) – and was thus termed the IRQ domain. A bioinformatics search resulted in a list of six additional proteins, with a high level of conservation in the central and flanking regions of the domain, designated IRQ1-6. The newly identified IRQs along with the aforementioned NETs were named the IRQ family. Rab proteins are part of the Ras superfamily of small guanosine triphosphatases (GTPases), which function as GDP/GTP regulated molecular switches and are widely known for their involvement in membrane trafficking; their role is to recruit effectors (e.g., motor proteins and tethering factors) that facilitate downstream membrane trafficking events. It has been shown in Arabidopsis thaliana that NET4A and NET4B interact with members of the RabG3 subfamily of Rab GTPases, RABG3A and RABG3F, and that the IRQ domain is responsible for this interaction. Further work determined that these NETs are true Rab effector proteins through the IRQ domain, which raised the hypothesis that the IRQ domain is a novel plant Rab interaction motif and that other IRQ domain-containing proteins may also be Rab effectors. Rab GTPases of the RabG subfamily, also referred to as Rab7 GTPases, are involved in a vesicular trafficking pathway to the vacuole, which starts from the trans-Golgi network (TGN) to the pre-vacuolar compartments/multivesicular bodies (PVCs/MVBs) and ends at the vacuole, with the sequential activation of GTPases Rab5 (corresponding to RabF subfamily) and Rab7. This thesis focussed on the functional characterisation of two members of the IRQ protein family in Arabidopsis, IRQ2 and IRQ3, as potential actin-binding proteins recruited by RabG3 GTPases to mediate vacuole-related membrane trafficking events. The experiments performed in this thesis demonstrated that IRQ2 and IRQ3 interact with RabG3 GTPases, RABG3A and RABG3F, similarly to what was observed for the NET4A and NET4B proteins, and it was possible to prove, at least in part, that this interaction is dependent on the IRQ domain. However, the interaction domain of these IRQs with Rab GTPases appears to include other elements along the proteins, as studies involving the N-terminal region seemed to demonstrate. In addition to these results, it was also observed that the IRQ2 and IRQ3 proteins are distributed in distinct patterns in the plant cell, which may be related to these proteins binding different forms of actin; however, this aspect requires additional experimental confirmation.
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spelling Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeletonCitoesqueleto de actinaproteínas de ligação à actinatráfego de membranaRab GTPasesvacúoloActin cytoskeletonActin-binding proteinsmembrane traffickingvacuoleDomínio/Área Científica::Ciências Naturais::Ciências BiológicasThe spatiotemporal organisation of cells is essential for the correct performance of organisms, which implies a complex and highly regulated system. In eukaryotic cells, this aspect is of particular importance, with the cytoskeleton playing a key role as a determinant of cell architecture and function. Regarding plant cells, the actin cytoskeleton is involved in most developmental processes, regulating membrane trafficking, organelle movement, morphogenesis and the establishment and maintenance of cell polarity. The dynamics of the actin cytoskeleton are regulated by numerous actin-binding proteins (ABPs), that modulate the polymerisation/depolymerisation of filaments, mediate the transport of cellular components, contribute to the formation of higher-order actin structures, and serve as a bridge between the actin cytoskeleton and membrane structures in the cell. The NETWORKED (NET) proteins are a recently characterised, plant-specific superfamily of actin-binding proteins that serve as interfaces between the actin cytoskeleton and various cell membranes. Some members (NET3A, NET3C; NET4A, NET4B) share a conserved domain in the C-terminal region that has at its centre three conserved amino acids – isoleucine (I), arginine (R) and glutamine (Q) – and was thus termed the IRQ domain. A bioinformatics search resulted in a list of six additional proteins, with a high level of conservation in the central and flanking regions of the domain, designated IRQ1-6. The newly identified IRQs along with the aforementioned NETs were named the IRQ family. Rab proteins are part of the Ras superfamily of small guanosine triphosphatases (GTPases), which function as GDP/GTP regulated molecular switches and are widely known for their involvement in membrane trafficking; their role is to recruit effectors (e.g., motor proteins and tethering factors) that facilitate downstream membrane trafficking events. It has been shown in Arabidopsis thaliana that NET4A and NET4B interact with members of the RabG3 subfamily of Rab GTPases, RABG3A and RABG3F, and that the IRQ domain is responsible for this interaction. Further work determined that these NETs are true Rab effector proteins through the IRQ domain, which raised the hypothesis that the IRQ domain is a novel plant Rab interaction motif and that other IRQ domain-containing proteins may also be Rab effectors. Rab GTPases of the RabG subfamily, also referred to as Rab7 GTPases, are involved in a vesicular trafficking pathway to the vacuole, which starts from the trans-Golgi network (TGN) to the pre-vacuolar compartments/multivesicular bodies (PVCs/MVBs) and ends at the vacuole, with the sequential activation of GTPases Rab5 (corresponding to RabF subfamily) and Rab7. This thesis focussed on the functional characterisation of two members of the IRQ protein family in Arabidopsis, IRQ2 and IRQ3, as potential actin-binding proteins recruited by RabG3 GTPases to mediate vacuole-related membrane trafficking events. The experiments performed in this thesis demonstrated that IRQ2 and IRQ3 interact with RabG3 GTPases, RABG3A and RABG3F, similarly to what was observed for the NET4A and NET4B proteins, and it was possible to prove, at least in part, that this interaction is dependent on the IRQ domain. However, the interaction domain of these IRQs with Rab GTPases appears to include other elements along the proteins, as studies involving the N-terminal region seemed to demonstrate. In addition to these results, it was also observed that the IRQ2 and IRQ3 proteins are distributed in distinct patterns in the plant cell, which may be related to these proteins binding different forms of actin; however, this aspect requires additional experimental confirmation.Malhó, Rui Manuel dos SantosHussey, Patrick J.Repositório da Universidade de LisboaBraga, Teresa2023-01-01T01:31:43Z2021-122021-072021-12-01T00:00:00Zdoctoral thesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10451/53670TID:101579489enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-11-20T18:15:15Zoai:repositorio.ul.pt:10451/53670Portal AgregadorONGhttps://www.rcaap.pt/oai/openairemluisa.alvim@gmail.comopendoar:71602024-11-20T18:15:15Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeleton
title Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeleton
spellingShingle Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeleton
Braga, Teresa
Citoesqueleto de actina
proteínas de ligação à actina
tráfego de membrana
Rab GTPases
vacúolo
Actin cytoskeleton
Actin-binding proteins
membrane trafficking
vacuole
Domínio/Área Científica::Ciências Naturais::Ciências Biológicas
title_short Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeleton
title_full Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeleton
title_fullStr Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeleton
title_full_unstemmed Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeleton
title_sort Characterisation of two members of the Arabidopsis thaliana IRQ family of proteins : a nexus for membrane trafficking and the actin cytoskeleton
author Braga, Teresa
author_facet Braga, Teresa
author_role author
dc.contributor.none.fl_str_mv Malhó, Rui Manuel dos Santos
Hussey, Patrick J.
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Braga, Teresa
dc.subject.por.fl_str_mv Citoesqueleto de actina
proteínas de ligação à actina
tráfego de membrana
Rab GTPases
vacúolo
Actin cytoskeleton
Actin-binding proteins
membrane trafficking
vacuole
Domínio/Área Científica::Ciências Naturais::Ciências Biológicas
topic Citoesqueleto de actina
proteínas de ligação à actina
tráfego de membrana
Rab GTPases
vacúolo
Actin cytoskeleton
Actin-binding proteins
membrane trafficking
vacuole
Domínio/Área Científica::Ciências Naturais::Ciências Biológicas
description The spatiotemporal organisation of cells is essential for the correct performance of organisms, which implies a complex and highly regulated system. In eukaryotic cells, this aspect is of particular importance, with the cytoskeleton playing a key role as a determinant of cell architecture and function. Regarding plant cells, the actin cytoskeleton is involved in most developmental processes, regulating membrane trafficking, organelle movement, morphogenesis and the establishment and maintenance of cell polarity. The dynamics of the actin cytoskeleton are regulated by numerous actin-binding proteins (ABPs), that modulate the polymerisation/depolymerisation of filaments, mediate the transport of cellular components, contribute to the formation of higher-order actin structures, and serve as a bridge between the actin cytoskeleton and membrane structures in the cell. The NETWORKED (NET) proteins are a recently characterised, plant-specific superfamily of actin-binding proteins that serve as interfaces between the actin cytoskeleton and various cell membranes. Some members (NET3A, NET3C; NET4A, NET4B) share a conserved domain in the C-terminal region that has at its centre three conserved amino acids – isoleucine (I), arginine (R) and glutamine (Q) – and was thus termed the IRQ domain. A bioinformatics search resulted in a list of six additional proteins, with a high level of conservation in the central and flanking regions of the domain, designated IRQ1-6. The newly identified IRQs along with the aforementioned NETs were named the IRQ family. Rab proteins are part of the Ras superfamily of small guanosine triphosphatases (GTPases), which function as GDP/GTP regulated molecular switches and are widely known for their involvement in membrane trafficking; their role is to recruit effectors (e.g., motor proteins and tethering factors) that facilitate downstream membrane trafficking events. It has been shown in Arabidopsis thaliana that NET4A and NET4B interact with members of the RabG3 subfamily of Rab GTPases, RABG3A and RABG3F, and that the IRQ domain is responsible for this interaction. Further work determined that these NETs are true Rab effector proteins through the IRQ domain, which raised the hypothesis that the IRQ domain is a novel plant Rab interaction motif and that other IRQ domain-containing proteins may also be Rab effectors. Rab GTPases of the RabG subfamily, also referred to as Rab7 GTPases, are involved in a vesicular trafficking pathway to the vacuole, which starts from the trans-Golgi network (TGN) to the pre-vacuolar compartments/multivesicular bodies (PVCs/MVBs) and ends at the vacuole, with the sequential activation of GTPases Rab5 (corresponding to RabF subfamily) and Rab7. This thesis focussed on the functional characterisation of two members of the IRQ protein family in Arabidopsis, IRQ2 and IRQ3, as potential actin-binding proteins recruited by RabG3 GTPases to mediate vacuole-related membrane trafficking events. The experiments performed in this thesis demonstrated that IRQ2 and IRQ3 interact with RabG3 GTPases, RABG3A and RABG3F, similarly to what was observed for the NET4A and NET4B proteins, and it was possible to prove, at least in part, that this interaction is dependent on the IRQ domain. However, the interaction domain of these IRQs with Rab GTPases appears to include other elements along the proteins, as studies involving the N-terminal region seemed to demonstrate. In addition to these results, it was also observed that the IRQ2 and IRQ3 proteins are distributed in distinct patterns in the plant cell, which may be related to these proteins binding different forms of actin; however, this aspect requires additional experimental confirmation.
publishDate 2021
dc.date.none.fl_str_mv 2021-12
2021-07
2021-12-01T00:00:00Z
2023-01-01T01:31:43Z
dc.type.driver.fl_str_mv doctoral thesis
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10451/53670
TID:101579489
url http://hdl.handle.net/10451/53670
identifier_str_mv TID:101579489
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv mluisa.alvim@gmail.com
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