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

Abstract

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.

Contrasting patterns in the evolution of the Rab GTPase family in Archaeplastida

“…Hoepflinger et al noticed the existence of Rab5-like proteins with the same modification in some members of the Alveolata group (e.g. the apicomplexan Plasmodium falciparum) [41]. However, their phylogenetic analysis failed to provide evidence for the common origin of the myristoylated/palmitolyated Rab5-like paralogs in Chloroplastida and Alveolata, and this negative result is consistent with our own, even broader phylogenies of the Rab5-related group (unpublished data).…”

“…It was previously claimed that RabF1 is missing also from O. lucimarinus [41], but we found in this species an obvious ortholog that also possess the characteristic N-terminal extension (Tab. S1 and Fig.…”

“…In addition, it was recently suggested that the group of Rab5-like sequences known from some Chloroplastida and typified by the A. thaliana gene RabF1 (also called ARA6 [40]) may represent a paralog that originated before the divergence of Archaeplastida [41]. Fig.…”

“…This group is characterized by the absence of a C-terminal geranylgeranylated tail, which is functionally replaced by an N-terminal extension modified by myristoylation and palmitoylation [40,41]. Hoepflinger et al noticed the existence of Rab5-like proteins with the same modification in some members of the Alveolata group (e.g.…”

“…The failure of Hoepflinger et al [41] to detect this ortholog may relate to the fact that the respective gene model is incomplete in the official genome annotation release for this species. We were also able to correct the gene model for the protein sequence EFN55859 from Chlorella variabilis that was noticed by Hoepflinger et al [41] as lacking the N-terminal region; the revised sequence has the N-terminal extension typical for all RabF1 proteins (Fig. S3).…”