Molecular and biochemical analysis of the first ARA6 homologue, a RAB5 GTPase, from green algae

• Published in: Journal of experimental botany 2013-12, Vol.64 (18), p.5553-5568
• Main Authors: Hoepflinger, Marion C, Geretschlaeger, Anja, Sommer, Aniela, Hoeftberger, Margit, Nishiyama, Tomoaki, Sakayama, Hidetoshi, Hammerl, Peter, Tenhaken, Raimund, Ueda, Takashi, Foissner, Ilse
• Format: Article
• Language: English
• Subjects: Algae; Amino Acid Sequence; Animals; Antibodies; Arabidopsis Proteins - immunology; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Biological and medical sciences; Bryopsida; Cell Membrane - metabolism; Cell membranes; Chara; Chara - enzymology; Chara - genetics; Chara australis; Characeae; Chlorophycota; electron microscopy; embryophytes; Endosomes; Endosomes - metabolism; Epidermal cells; epitopes; fluorescent antibody technique; Fluorescent antibody techniques; Fundamental and applied biological sciences. Psychology; guanosinetriphosphatase; leaves; Mice; Molecular Sequence Data; molecular weight; Multivesicular Bodies - metabolism; mutation; Nicotiana - genetics; Nicotiana benthamiana; Organelles; Phylogeny; physiological transport; Plant cells; Plant Epidermis - genetics; Plant Leaves - genetics; Plant Leaves - metabolism; Plant physiology and development; Plant Proteins - genetics; Plant Proteins - metabolism; Plants; Plants, Genetically Modified - genetics; plasma membrane; Point Mutation; polypeptides; proteins; rab GTP-Binding Proteins - genetics; rab GTP-Binding Proteins - immunology; rab GTP-Binding Proteins - metabolism; RESEARCH PAPER; Sequence Homology, Amino Acid; yeasts
• ISSN: 0022-0957; 1460-2431
• DOI: 10.1093/jxb/ert322

RAB5 GTPases are important regulators of endosomal membrane traffic in yeast, plants, and animals. A specific subgroup of this family, the ARA6 group, has been described in land plants including bryophytes, lycophytes, and flowering plants. Here, we report on the isolation of an ARA6 homologue in a green alga. CaARA6 (CaRABF1) from Chara australis, a member of the Characeae that is a close relative of land plants, encodes a polypeptide of 237 aa with a calculated molecular mass of 25.4kDa, which is highly similar to ARA6 members from Arabidopsis thaliana and other land plants and has GTPase activity. When expressed in Nicotiana benthamiana leaf epidermal cells, fluorescently tagged CaARA6 labelled organelles with diameters between 0.2 and 1.2 µm, which co-localized with fluorescently tagged AtARA6 known to be present on multivesicular endosomes. Mutations in the membrane-anchoring and GTP-binding sites altered the localization of CaARA6 comparable to that of A. thaliana ARA6 (RABF1). In characean internodal cells, confocal immunofluorescence and immunogold electron microscopy with antibodies against AtARA6 and CaARA6 revealed ARA6 epitopes not only at multivesicular endosomes but also at the plasma membrane, including convoluted domains (charasomes), and at the trans-Golgi network. Our findings demonstrate that ARA6-like proteins have a more ancient origin than previously thought. They indicate further that ARA6-like proteins could have different functions in spite of the high similarity between characean algae and flowering plants.