Journal Article

OsGAP1 Functions as a Positive Regulator of OsRab11-mediated TGN to PM or Vacuole Trafficking

Jae Bok Heo, Hee Sun Rho, Se Won Kim, Sung Min Hwang, Hyun Jin Kwon, Min Yeop Nahm, Woo Young Bang and Jeong Dong Bahk

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 12, pages 2005-2018
Published in print December 2005 | ISSN: 0032-0781
Published online December 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci215
OsGAP1 Functions as a Positive Regulator of OsRab11-mediated TGN to PM or Vacuole Trafficking

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase–green fluorescent protein (GFP) and Ca2+-ATPase8–GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)–GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11.

Keywords: OsGAP1; OsRab11; Pre-vacuolar compartment; Trans-Golgi network; Vesicular trafficking; AALP, Arabidopsis aleurain-like protein; ACA8, Ca2+-ATPase8; BFA, brefeldin A; ER, endoplasmic reticulum; EST, expressed sequence tag; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; ORF, open reading frame; OsGAP1, Oryza sativa GTPase-activating protein 1; OsGDI3, Oryza sativa GDP dissociation inhibitor 3; PEI, poly(ethyleneimine); PM, plasma membrane; PVC, pre-vacuolar compartment; RFP, red fluorescent protein; TGN, trans-Golgi network; TLC, thin-layer chromatography

Journal Article.  9607 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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