Degradation of structure variants of boric acid channels through the endoplasmic reticulum-associated degradation pathway in Arabidopsis.

Abstract

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 these boric acid channels to their localization and identified critical leucine residues at the boundary of the N-terminal cytosolic region and the first 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 a 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 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 the folding and PM targeting of boric acid channels, and that ERAD operates 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.