OsGAP1 functions as a positive regulator of OsRab11-mediated TGN to PM or vacuole trafficking.
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.
Heo
Jae Bok
JB
Division of Applied Life Sciences, Graduate School of Gyeongsang National University, Jinju 660-701, Korea.
Rho
Hee Sun
HS
Kim
Se Won
SW
Hwang
Sung Min
SM
Kwon
Hyun Jin
HJ
Nahm
Min Yeop
MY
Bang
Woo Young
WY
Bahk
Jeong Dong
JD
D000409:Alanine / Q000032:analysis; D000595:Amino Acid Sequence; D001120:Arginine / Q000032:analysis; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D018744:DNA, Plant / Q000032:analysis / Q000302:isolation & purification; D020690:GTPase-Activating Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000502:physiology; D006868:Hydrolysis; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D021601:trans-Golgi Network / Q000737:chemistry / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D000409:Alanine; D001120:Arginine; D018744:DNA, Plant; D020690:GTPase-Activating Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D010940:Plant Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins