Poster: Protein targeting & vesicular trafficking

Abs # 643: A plant GAP homologue, OsGAP1, modulates the vesicular trafficking to plasma membrane in vivo

Presenter:	Bahk, Jeong D., jdbahk@nongae.gsnu.ac.kr
Authors	Bahk, Jeong D. (A) (B)  Heo, Jae B. (A)   Hwang, Seong M. (A)   Rho, Hee S. (A)   Kim, Se W. (A)   Jeong, Eun J. (A)
Affiliations:	(A): Div. of Applied Life Sci, Graduate School of Gyeongsang Natl. Univ. (B): Agricultural Plant Stress Research Center, Chonnam Natl. Univ.

Small G-proteins of the YPT/Rab family play a key role in regulating the vesicular transport between intracellular compartments. Their intrinsic rates of GTP hydrolysis are very slow but accelerated by specific GTPase activating proteins (GAPs) which switch them from the active to the inactive state. Here we report on the identification and characterization of a novel plant Rab specific GAP named as OsGAP1, whose efficient substrate is an OsRab11 homologous to the mammalian Rab11, essential for the trans-Golgi network (TGN)-to-plasma membrane (PM) trafficking. OsGAP1 contains several conserved motifs called to 'GAP domains' for the GTPase activity. Especially, substitution of alanines for two invariant arginines in the catalytic domains of OsGAP1 resulted in significant inhibition of GAP activity. Yeast two-hybrid and GST-pull down assays revealed that OsGAP1 might function in a homo-dimeric form. In vivo targeting experiments demonstrated that OsGAP1 was localized on the TGN and/or the prevacuolar compartment. Using green fluorescent protein (GFP)-OsRab11, we demonstrated that OsGAP1 facilitates the recycling of OsRab11 to be catalyzed by OsGDI3. The expressed OsGAP1 (R385A) mutants in Arabidopsis protoplasts inhibited the trafficking of two cargo proteins, H⁺-ATPase-GFP and Ca²⁺-ATPase8-GFP, destined for the plasma membrane, and accumulated them to the Golgi apparatus. Furthermore, overexpression of OsRab11 relieved the inhibitory effect of OsGAP1 (R385A) mutant. These results suggest that OsGAP1 is involved in the cycling of OsRab11 at the early stage, and then modulates the vesicular trafficking to the plasma membrane in plant cell.[This work was supported by the BK21 project at Gyeongsang National University and in part by APSRC at Chonnam National University]