Characterization and subcellular localization of a small GTP-binding protein (Ara-4) fromArabidopsis: conditional expression under control of the promoter of the gene for heat-shock protein HSP81-1

Abstract

Small GTP-binding proteins belonging to the rab/YPT family play key roles at various steps in intracellular transport pathways in yeast and mammalian cells. Many members of rab/YPT family have been isolated from plants to date. However, detailed information about the localization and function of the gene products remains limited, even though intracellular transport is likely to be involved in important phenomena such as cell elongation, transport of storage proteins, determination and maintenance of cell polarity and intercellular signal transduction. We have attempted to establish transgenic Arabidopsis plants that overexpress ARA-4, a rab/YPT homologue in order to analyze the function and the localization of the gene product. For overexpression and also for regulation of the expression of this gene, the promoter of the gene for HSP81-1 was employed to drive the transcription of ARA-4 in transgenic plants. The response of the introduced genes to heat shock was analyzed. Upon heat-shock treatment, the ARA-4 gene was efficiently transcribed and translated. The induction of ARA-4 by heat shock was transient, and at least two distinct forms of this protein were found in membrane and cytosolic fractions from transgenic plants. Prolonged incubation after heat shock reduced the amount of the cytosolic form of the induced protein, and the cytosolic form of the protein thus probably represents the unprocessed precursor. Using transgenic plants, we determined the subcellular localization of the product of ARA-4. The protein was predominantly localized on Golgi-derived vesicles, Golgi cisternae and the trans-Golgi network.

BEX5/RabA1b Regulates trans-Golgi Network-to-Plasma Membrane Protein Trafficking in Arabidopsis

“…In plants, members of RabA GTPases have also been associated with post-Golgi endomembranes (Ueda et al, 1996;Inaba et al, 2002;Preuss et al, 2004;Chow et al, 2008). So far, there is no information about the function of Arabidopsis RabA1 proteins.…”

“…The best-represented subfamily is the RabA GTPases (Rutherford and Moore, 2002;Vernoud et al, 2003;Bassham et al, 2008). Characterization of dominant negative (DN) and constitutive active (CA) mutants and localization studies showed that RabA GTPase members play a role in post-Golgi protein trafficking (Ueda et al, 1996;Preuss et al, 2004;Chow et al, 2008).…”

Rab-A2 and Rab-A3 GTPases Define atrans-Golgi Endosomal Membrane Domain inArabidopsisThat Contributes Substantially to the Cell Plate

“…A member of the Arabidopsis Rab-A4 subclass, RAB-A4 b , has been implicated in tip growth in root hairs via its interaction with two phosphatidylinositol-4-kinases that together are essential for root hair morphogenesis (Preuss et al, 2006). RAB-A4 b targets yellow fluorescent protein (YFP) to membranes near the trans-face of the Golgi stacks at the tips of elongating root hairs (Preuss et al, 2004), and RAB-A5 c (ARA4) has been immunolocalized to Golgi-associated vesicles in pollen (Ueda et al, 1996).…”

Rab11 GTPase-Regulated Membrane Trafficking Is Crucial for Tip-Focused Pollen Tube Growth in Tobacco

“…In plants, green fluorescent protein (GFP)-tagged Rab11 homologs from pea (Pisum sativum), Pra-2 and Pra-3, have been localized to Golgi bodies and endosomes, respectively, when expressed in tobacco cells (Inaba et al, 2002), whereas GFP-Pra2 localized to the endoplasmic reticulum when expressed in Arabidopsis (Kang et al, 2001). Cytoimmunodetection using a monoclonal antibody specific for an Arabidopsis Rab11, ARA4 (AtRABA5c), revealed a Golgi and post-Golgi vesicle localization (Ueda et al, 1996). Another Arabidopsis Rab11 homolog, AtRABA4b, cofractionated with a non-trans-Golgi network membrane fraction (Preuss et al, 2004).…”