Pratelli R, Guerra DD, Yu S, Wogulis M, Kraft E, Frommer WB, Callis J, Pilot G

Amino acids serve as transport forms for organic nitrogen in the plant and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, over-expression of GLUTAMINE DUMPER 1 (GDU1) in Arabidopsis thaliana led to increased amino acid export. To gain insight ...

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into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS of GDU 2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by GST pull-down, in vitro ubiquitination, and in planta co-immunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant, log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial miRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

Date: Jan. 30, 2012

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