Transport-coupled ubiquitination of the borate transporter BOR1 for its boron-dependent degradation

Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.

The borate transporter BOR1 senses the boron concentration through its borate transport activity for K63-linked polyubiquitination and subsequent degradation.     

Proton magnetic resonance spectra of D-gluco-oligosaccharides and D-glucans

The p.m.r. spectra of some D-gluco-oligosaccharides and D-glucans in deuterium oxide were studied with respect to the anomeric proton. In (1→2)-linked glucobioses, the effect of change in configuration of the hydroxyl group at C-1 on the chemical shifts of the glycosidic proton is noted. Equilibrium mixtures of (1→2)-linked glucobioses contained more α-anomer than did the other examples, despite the cis configuration of substituents at C-1 and C-2. Some D-glucans were investigated with regard to the degree of branching, although solubility was a limitation.     

Enzymatic synthesis of β-D-Gal-(1→3)-[β-D-GlcNAc-(1→6)]-α-D-GalNAc-OC6H4NO2-p as a carbohydrate unit of mucin-type 2 core

We have established a synthetic method for obtaining β-D-Gal-(1→3)-[β-D-GlcNAc-(1→6)]-α-D-GalNAc-OC6H4NO2 -p (1), which is a carbohydrate unit of mucin-type 2 core. A β-N-acetyl-D-hexosaminidase from Nocardia orientalis catalyzed the synthesis of the desired compound 1 with its isomers β-D-GalNAc-(1→6)-β-D-Gal-(1→3)-α-D-GalNAc-OC6H4NO2-p (2) β-D-GlcNAc-(1→3)-β-D-Glc-(1→3)-α-D-GalNAc-OC6H4NO2-p (3) through N-acetylglucosaminyl transfer from N,N′-diacetylchitobiose and β-D-Gal-(1→3)-α-D-GalNAc-OC6H4NO2-p. The enzyme formed the trisaccharides 1, 2, and 3 in 14% overall yield based on β-D-Gal-(1→3)-α-D-GalNAc-OC6H4NO2-p as an acceptor substrate, and in the ratio of 44:32:24. In this way, N-acetylglucosaminyl transfer favored O-6 of the acceptor rather than O-6′, and occurred to a lesser extent at O-3′. This reaction was efficient enough to allow a one-pot preparation of the desired carbohydrate unit of mucin-type 2 core. When β-D-Gal-(1→3)-β-D-GalNAc-OC6H4NO2-p was used as an acceptor, the enzyme also synthesized three kinds of trisaccharides in the same regioselectivity with respect to O-6 and O-6′ versus O-3′ of the acceptor.