Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae

• Published in: Traffic (Copenhagen, Denmark) Denmark), 2015-05, Vol.16 (5), p.534-554
• Main Authors: Hoepflinger, Marion C., Geretschlaeger, Anja, Sommer, Aniela, Hoeftberger, Margit, Hametner, Christina, Ueda, Takashi, Foissner, Ilse
• Format: Article
• Language: English
• Subjects: Algae; Algal Proteins - genetics; Algal Proteins - metabolism; Amino Acid Sequence; ARA7; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Binding sites; Chara australis; Characeae - enzymology; Characeae - genetics; Electrophoresis, Polyacrylamide Gel; endosomal trafficking; Molecular Sequence Data; multivesicular endosome; Phylogeny; Plant Leaves - enzymology; Plant Leaves - genetics; Point Mutation; rab GTP-Binding Proteins - genetics; rab GTP-Binding Proteins - metabolism; rab5 GTP-Binding Proteins - genetics; rab5 GTP-Binding Proteins - metabolism; RAB5 GTPase; RABF2; Sequence Alignment; Sequence Homology, Amino Acid
• ISSN: 1398-9219; 1600-0854
• DOI: 10.1111/tra.12267

This is the first detailed investigation of a conventional RAB5 GTPase (CaARA7/CaRABF2) in a green alga closely related to land plants (Chara australis; Charales). CaARA7 is highly similar to other RAB5 proteins and has intrinsic GTPase activity. It is involved in endosomal membrane trafficking as suggested by localization experiments (immunolabelling of internodal cells using anti‐CaARA7 as well as transient expression of different fluorescently tagged RAB5s in tobacco) and by protein mutations in membrane anchoring and GTP binding sites. RAB5 GTPases are important regulators of endosomal membrane traffic. Among them Arabidopsis thaliana ARA7/RABF2b is highly conserved and homologues are present in fungal, animal and plant kingdoms. In land plants ARA7 and its homologues are involved in endocytosis and transport towards the vacuole. Here we report on the isolation of an ARA7 homologue (CaARA7/CaRABF2) in the highly evolved characean green alga Chara australis. It encodes a polypeptide of 202 amino acids with a calculated molecular mass of 22.2 kDa and intrinsic GTPase activity. Immunolabelling of internodal cells with a specific antibody reveals CaARA7 epitopes at multivesicular endosomes (MVEs) and at MVE‐containing wortmannin (WM) compartments. When transiently expressed in epidermal cells of Nicotiana benthamiana leaves, fluorescently tagged CaARA7 localizes to small organelles (putative MVEs) and WM compartments, and partially colocalizes with AtARA7 and CaARA6, a plant specific RABF1 GTPase. Mutations in membrane anchoring and GTP binding sites alter localization of CaARA7 and affect GTPase activity, respectively. This first detailed study of a conventional RAB5 GTPase in green algae demonstrates that CaARA7 is similar to RAB5 GTPases from land plants and other organisms and shows conserved structure and localization.