ADP-ribosylation factor 1 of Arabidopsis plays a critical role in intracellular trafficking and maintenance of endoplasmic reticulum morphology in Arabidopsis.
ADP-ribosylation factors (Arf), a family of small GTP-binding proteins, play important roles in intracellular trafficking in animal and yeast cells. Here, we investigated the roles of two Arf homologs, Arf1 and Arf3 of Arabidopsis, in intracellular trafficking in plant cells. We generated dominant negative mutant forms of Arf 1 and Arf3 and examined their effect on trafficking of reporter proteins in protoplasts. Arf1[T31N] inhibited trafficking of H(+)-ATPase:green fluorescent protein (GFP) and sialyltransferase (ST):GFP to the plasma membrane and the Golgi apparatus. In addition, Arf1[T31N] caused relocalization of the Golgi reporter protein ST:GFP to the endoplasmic reticulum (ER). In protoplasts expressing Arf1[T31N], ST:red fluorescent protein remained in the ER, whereas H(+)-ATPase:GFP was mistargeted to another organelle. Also, expression of Arf1[T31N] in protoplasts resulted in profound changes in the morphology of the ER. The treatment of protoplasts with brefeldin A had exactly the same effect as Arf1[T31N] on various intracellular trafficking pathways. In contrast, Arf3[T31N] did not affect trafficking of any of these reporter proteins. Inhibition experiments using mutants with various domains swapped between Arf1 and Arf3 revealed that the N-terminal domain is interchangeable for trafficking inhibition. However, in addition to the T31N mutation, motifs in domains II, III, and IV of Arf1 were necessary for inhibition of trafficking of H(+)-ATPase:GFP. Together, these results strongly suggest that Arf1 plays a role in the intracellular trafficking of cargo proteins in Arabidopsis, and that Arf1 functions through a brefeldin A-sensitive factor.
Lee
Mi Hee
MH
Center for Plant Intracellular Trafficking, Pohang University of Science and Technology, Pohang, 790-784, Korea.
Min
Myung Ki
MK
Lee
Yong Jik
YJ
Jin
Jing Bo
JB
Shin
Dong Han
DH
Kim
Dae Heon
DH
Lee
Kwang-Hee
KH
Hwang
Inhwan
I
D020823:ADP-Ribosylation Factor 1 / Q000502:physiology*; D020727:ADP-Ribosylation Factors / Q000502:physiology; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000502:physiology*; D029681:Arabidopsis Proteins; D001692:Biological Transport / Q000502:physiology; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D017136:Ion Transport / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D030821:Plants, Genetically Modified; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D011523:Protoplasts / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000037:antagonists & inhibitors
D029681:Arabidopsis Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D012799:Sialyltransferases; D006180:Proton-Translocating ATPases; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C492130:ADP-ribosylation factor 3 protein, Arabidopsis