In vitro and in vivo interaction of AtRma2 E3 ubiquitin ligase and auxin binding protein 1

Biochem Biophys Res Commun. 2010 Mar 12;393(3):492-7. doi: 10.1016/j.bbrc.2010.02.032. Epub 2010 Feb 10.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Cysteine Proteinase Inhibitors / pharmacology
  • Leupeptins / pharmacology
  • Nicotiana / cytology
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism*
  • Two-Hybrid System Techniques
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination*

Substances

  • Arabidopsis Proteins
  • Cysteine Proteinase Inhibitors
  • Leupeptins
  • Plant Proteins
  • Receptors, Cell Surface
  • auxin-binding protein 1
  • Rma2 protein, Arabidopsis
  • Ubiquitin-Protein Ligases
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde