In vitro and in vivo interaction of AtRma2 E3 ubiquitin ligase and auxin binding protein 1.
E3 ubiquitin (Ub) ligases play diverse roles in cellular regulation in eukaryotes. Three homologous AtRmas (AtRma1, AtRma2, and AtRma3) were recently identified as ER-localized Arabidopsis homologs of human RING membrane-anchor E3 Ub ligase. Here, auxin binding protein 1 (ABP1), one of the auxin receptors in Arabidopsis, was identified as a potential substrate of AtRma2 through a yeast two-hybrid assay. An in vitro pull-down assay confirmed the interaction of full-length AtRma2 with ABP1. AtRma2 was transiently expressed in tobacco (Nicotiana benthamiana) plants through an Agrobacterium-mediated infiltration method and bound ABP1 in vivo. In vitro ubiquitination assays revealed that bacterially-expressed AtRma2 ubiquitinated ABP1. ABP1 was poly-ubiquitinated in tobacco cells and its stability was significantly increased in the presence of MG132, a 26S proteasome inhibitor. This suggests that ABP1 is controlled by the Ub/26S proteasome system. Therefore, AtRma2 is likely involved in the cellular regulation of ABP1 expression levels.
Department of Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.
Ora
O
Son
Seok Keun
SK
Cho
Soo Jin
SJ
Kim
Woo Taek
WT
Kim
D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D029681:Arabidopsis Proteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D010940:Plant Proteins; D011956:Receptors, Cell Surface; C083846:auxin-binding protein 1; C548549:Rma2 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde