Degradation of Structure Variants of Boric Acid Channels through ERAD Pathway in Arabidopsis.

The nodulin 26-like intrinsic protein NIP5;1 is a boric acid channel localized in the plasma membrane (PM) for efficient uptake of B in roots of Arabidopsis thaliana under low B conditions. NIP6;1 is the closest paralog of NIP5;1 and is responsible for B distribution to young tissues in shoots. In the present study, we analyzed the contribution of the N-terminal cytosolic region of the boric acid channels in their localization and identified critical leucine residues at the boundary of the N-terminal cytosolic region and the 1st transmembrane helix (L76 of NIP5;1 and L78 of NIP6;1). Substitution of the leucine residues by an alanine residue but not by phenylalanine in GFP-NIP5;1 and GFP-NIP6;1 reduced protein levels in the PM in nip5;1 mutant background. The GFP-NIP5;1 L76A was observed in the PM when expressed in WT Col-0 background, suggesting that the defective variant can be transported to the PM as oligomers containing endogenous NIP5;1. When proteasome activity was inhibited by MG132, GFP signal derived from GFP-NIP5;1 L76A and GFP-NIP6;1 L78A was accumulated in the cytoplasm. These results suggest that NIP5;1 L76A and NIP6;1 L78A were subjected to endoplasmic reticulum (ER)-associated degradation (ERAD). These findings indicate that the conserved leucine or phenylalanine is essential for folding and PM targeting of boric acid channels, and ERAD is operating to eliminate unfolded/misfolded boric acid channels in plant cells. GFP-NIP5;1 L76A and GFP-NIP6;1 L78A will be used as fluorescent markers for ERAD studies in plant cells.