Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana

Abstract

Ara6 of Arabidopsis thaliana is a novel member of the Rab/Ypt GTPase family with unique structural features. It resembles Rab5 GTPases best, but lacks a large part of the C-terminal hypervariable region and the cysteine motif, and instead harbors an extra stretch of amino acid residues containing myristoylation and palmitoylation sites at the N-terminus. Ara6 is tightly associated with membranes and is expressed constitutively. In contrast, the conventional Rab5 ortholog, Ara7, is highly expressed only in actively dividing cells. Examination of green fluorescent protein (GFP)-tagged proteins indicates that both Ara6 and Ara7 are distributed on a subpopulation of endosomes and suggests their roles in endosomal fusion. The endosomal localization of Ara6 requires N-terminal fatty acylation, nucleotide binding and the C-terminal amino acid sequence coordinately. Proteins similar to Ara6 are found only in higher plants and thus represent a novel class of Rab GTPases regulating endocytic function in a plant- specific manner.

The AP-3 adaptor complex is required for vacuolar function in Arabidopsis

“…During internalization, the endocytic tracer FM4-64 labels the EEs, subsequently it marks intracellular compartments and finally it reaches the vacuole and labels the vacuolar membrane, the tonoplast [28]. In the pat4-1 mutant after long treatment (3 h) with 4 µM FM4-64, the dye stained the ectopic intracellular accumulations of PIN1-GFP ( Figure 2C and 2D).…”

Rice SCAMP1 Defines Clathrin-Coated,trans-Golgi–Located Tubular-Vesicular Structures as an Early Endosome in Tobacco BY-2 Cells

“…In addition, the fluorescent dyes FM4-64 and FM1-43 have been successfully used to study the endocytic pathway in plant cells. After their insertion in the PM, these styryl dyes pass through endosomal compartments on their way to the tonoplast (Vida andEmr, 1995, Kim et al, 2001;Ueda et al, 2001;Emans et al, 2002;Tse et al, 2004;Dettmer et al, 2006). Therefore, colocalization of proteins with internalized FM4-64 may be used to judge whether a particular compartment labeled by a specific protein is a putative endosomal compartment in plant cells.…”

“…Therefore, colocalization of proteins with internalized FM4-64 may be used to judge whether a particular compartment labeled by a specific protein is a putative endosomal compartment in plant cells. Proteins localized to plant endosomes in this way include the Arabidopsis thaliana Rab GTPases ARA6 and ARA7 (Ueda et al, 2001(Ueda et al, , 2004, Arabidopsis PRA2 (SYP111) (Inaba et al, 2002), the small GTPase ARF1 in maize (Zea mays) (Baluska et al, 2002, the Arabidopsis GNOM protein and the auxin-efflux carrier PIN1 (Geldner et al, 2003), and vacuolar H þ -ATPase (Dettmer et al, 2006). However, in the great majority of these cases, the ultrastructural morphology of the compartments bearing these proteins was not given.…”

Evidence for a sorting endosome in Arabidopsis root cells

“…To date, only a few endosomal proteins have been described in plants, and endosome differentiation and functions are not well defined. The first plant proteins shown to associate with the membrane of endosomes were the Arabidopsis Rab GTPases AtRABF1 (also known as Ara6) and AtRABF2b (also known as Ara7) (Ueda et al, 2001(Ueda et al, , 2004. RABF1 and RABF2b are two of three Arabidopsis homologues of the mammalian small GTPase Rab5.…”

“…Rab GTPases are key regulators of vesicular transport and vesicle fusion (Zerial and McBride, 2001). In the protoplasts of Arabidopsis suspension-cultured cells, AtRABF1 and AtRABF2b were located to two distinct populations of endosomes, with some overlap (Ueda et al, 2001(Ueda et al, , 2004. By following internalization of the fluorescent lipophilic styryl dye FM4-64, AtRABF1 was localized mainly to a late type of endosome, whereas AtRABF2b was localized on early endosomes (Ueda et al, 2004).…”