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.     

Development of second generation merozoites of Leucocytozoon caulleryi in vitro

Sporozoites of Leucocytozoon caulleryi were inoculated into specific-pathogen-free (SPF) chickens intravenously. Fourteen days after inoculation, the infected blood, parasitized with second generation merozoites, was collected from the chickens. The blood was then suspended in SPF chicken serum or RPMI-1640 culture medium supplemented with 20% SPF chicken serum in petri dishes, which were cultured at 37 degrees C or 41 degrees C in a humidified atmosphere of 5% CO2. Second generation merozoites in parasitized, immature erythrocytes developed continuously through several substages and finally became micro- and macro-gametocytes after 6 days of incubation.     

Dependence of removal of sperm-specific proteins from Xenopus sperm nuclei on the phosphorylation state of nucleoplasmin

In Xenopus laevis , nucleoplasmin from fully grown oocytes is not highly phosphorylated, but is more extensively phosphorylated during oocyte maturation to retain this state until mid-blastula transition. Incubation of demembranated sperm with nucleoplasmin from oocytes or mature eggs revealed that egg nucleoplasmin is twice as potent as oocyte nucleoplasmin in removing sperm-specific basic proteins from chromatin (protamine-removing activity: PRA). Dephosphorylation of egg nucleoplasmin by alkaline phosphatase induced a remarkable decline of PRA in nucleoplasmin. Treatment of oocyte nucleoplasmin with cdc2 protein kinase induced an increase of the extent of phosphorylation, but to a level lower than that exhibited by egg nucleoplasmin, suggesting the involvement of other unspecified kinase(s) in phosphorylating nucleoplasmin during oocyte maturation. Incubation of sperm with cdc2 kinase induced selective phosphorylation of sperm-specific basic proteins, accompanied by their enhanced removal from sperm chromatin upon exposure to high-salt solutions. These results suggest that removal of sperm-specific basic proteins from sperm chromatin in fertilized eggs is facilitated by phosphorylation of both nucleoplasmin and sperm-specific basic proteins.