title: ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies.
abstract: Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ring-like structures (1-2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP-ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP-ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP-ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells.
authors.affiliation: School of Life Sciences, Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
authors.forename: Tianran
authors.initials: T
authors.lastname: Jia
authors.forename: Caiji
authors.initials: C
authors.lastname: Gao
authors.forename: Yong
authors.initials: Y
authors.lastname: Cui
authors.forename: Junqi
authors.initials: J
authors.lastname: Wang
authors.forename: Yu
authors.initials: Y
authors.lastname: Ding
authors.forename: Yi
authors.initials: Y
authors.lastname: Cai
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueda
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakano
authors.forename: Liwen
authors.initials: L
authors.lastname: Jiang
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D057146:Multivesicular Bodies / Q000201:enzymology* / Q000235:genetics; D020125:Mutation, Missense; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C495672:Ara7 protein, Arabidopsis; D020691:rab GTP-Binding Proteins
keywords: ARA7(Q69L); MVB enlargement; homotypic fusion; multivesicular body; pre-vacuolar compartment; transgenic Arabidopsis cells.