Modes of interaction between the Arabidopsis Rab protein, Ara4, and its putative regulator molecules revealed by a yeast expression system

Abstract

SummaryAra4, a member of the Rab/Ypt GTPase family derived from Arabidopsis thaliana, causes severe growth inhibition when expressed in several yeast ypt mutants. Mutational analysis of ARA4 indicated that the Ara4 protein titrates at least three factors in yeast, including the GDP dissociation inhibitor (GDI). The coexpression of AtGDI1 (Arabidopsis GDI) suppressed the growth defect caused by Ara4 in yeast ypt1, suggesting that Ara4 and AtGDI1 interact in yeast to compensate for the titration of yeast GDI. We screened an Arabidopsis cDNA library for other suppressors that may also interact with Ara4 physiologically. A novel suppressor, SAY1, encoded a hydrophilic protein with two putative coiled-coil regions, which showed partial similarity to the yeast Vps27 protein. To understand the structural requirements of Ara4 for interacting with these molecules, we examined whether AtGDI1 and SAY1 could suppress the growth defect of ypt1 caused by various mutant versions of ARA4. The results indicated that the interaction between Ara4 and AtGDI1 depends on the conserved C-terminal Cys-motif and Thr44 in the effector domain of Ara4. In contrast, neither of these motifs is necessary for the interaction between Say1 and Ara4. This approach provides a powerful method to dissect complex interactions between a GTPase and its regulators.

Identification and characterization of AtCASP, a plant transmembrane Golgi matrix protein

“…Golgi matrix proteins are thought to be important for maintenance of the structure of the Golgi apparatus, and may also be involved in tethering of transport vesicles through Rab GTPases (Barr, 1999;Moyer et al, 2001;Short et al, 2001;Valsdottir et al, 2001;Gillingham and Munro, 2003). The role of Rab GTPases is being unraveled in plants, and current data indicate roles of these proteins in various transport routes within the cell (Batoko et al, 2000;Ueda et al, 2000Ueda et al, , 2001Rutherford and Moore, 2002;Nahm et al, 2003;Bolte et al, 2004;Kotzer et al, 2004;Preuss et al, 2004). It therefore seems likely that further plant Golgi matrix proteins may exist that could specifically interact with different Rabs, but these have yet to be identified.…”

A Rab1 GTPase Is Required for Transport between the Endoplasmic Reticulum and Golgi Apparatus and for Normal Golgi Movement in Plants

“…In plants, these subclasses are based almost entirely on sequence comparisons, and whether they represent functional groups is not clear. In the absence of effective in vivo trafficking assays, information on plant Rab GTPase function has been derived from complementation and expression studies in yeast (Palme et al, 1992;Bednarek et al, 1994;Ueda et al, 2000), immunolocalization data (Ueda et al, 1996), and expression studies (Terryn et al, 1993;Nagano et al, 1995;Moore et al, 1997). Transgenic approaches suggest that disrupting Rab function produces various developmental and cellular anomalies, but they have not identified the in vivo trafficking roles of any plant Rab GTPase (Cheon et al, 1993;Bischoff et al, 1999).…”

A dominant negative mutant of Sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells

“…Some of them have been shown to complement related yeast mutants, indicating the functional conservation between plants and yeast (Bednarek et al, 1994;Cheon et al, 1993;d'Enfert et al, 1992;Fabry et al, 1995;Kim et al, 1996;Kim et al, 1997;Loraine et al, 1996;Park et al, 1994;Takeuchi et al, 1998). The use of yeast mutants has also led to the identi®cation of plant regulators of these GTPases and the understanding of their molecular interactions by virtue of the negative effects of their expression Ueda et al, 1996;Ueda et al, 1998;Ueda et al 2000). However, the physiological roles of these GTPases in plant cells remain to be elucidated.…”