Identification of the Domains Involved in Promotion of Silica Formation in Glassin, a Protein Occluded in Hexactinellid Sponge Biosilica, for Development of a Tag for Purification and Immobilization of Recombinant Proteins.

Glassin, a protein occluded in biosilica of the hexactinellid sponge Euplectela, promotes silica formation from silicic acid at room temperature and neutral pH and is characterized by its primary structure which consists of a tandem repeat carrying three distinct domains, histidine and aspartic acid-rich (HD) domain, proline-rich (P) domain, and histidine and threonine-rich (HT) domain. The present study aims to clarify the domain responsible for the promotion of silica formation and to demonstrate usefulness of glassin and its domain as a tag for purification and immobilization of recombinant proteins. When each domain was mixed with silicic acid at neutral pH, silica was formed with HD domain as well as glassin, or a single repeat, but not with P or HT domain. Neither of amino or carboxy-terminal half of HD domain induced silica formation. The amount of silica formed with HD domain was significantly lower than that of glassin or a single repeat. HD domain fused with HT domain raised the amount of silica formed, while a HD domain fused with P domain, a mixture of HD and P domains, or a mixture of HD and HT domains has little effect on the promotion of silica formation. Collectively, a minimum sequence for promotion of silica formation is HD domain, whose activity can be enhanced by HT domain through a topological effect. In addition, practicality of glassin and HD domain was demonstrated by fusion of these sequences to green fluorescent protein which was successfully purified with Ni affinity chromatography and immobilized on silica.

Polar Localization of the Borate Exporter BOR1 Requires AP2-Dependent Endocytosis.

Boron (B) is an essential element in plants but is toxic when it accumulates to high levels. In root cells of Arabidopsis (Arabidopsis thaliana), the borate exporter BOR1 is polarly localized in the plasma membrane toward the stele side for directional transport of B. Upon high-B supply, BOR1 is rapidly internalized and degraded in the vacuole. The polar localization and B-induced vacuolar sorting of BOR1 are mediated by endocytosis from the plasma membrane. To dissect the endocytic pathways mediating the polar localization and vacuolar sorting, we investigated the contribution of the clathrin adaptor protein, ADAPTOR PROTEIN2 (AP2) complex, to BOR1 trafficking. In the mutants lacking µ- or σ-subunits of the AP2 complex, the polar localization and constitutive endocytosis of BOR1 under low-B conditions were dramatically disturbed. A coimmunoprecipitation assay showed association of the AP2 complex with BOR1, while it was independent of YxxΦ sorting motifs, which are in a cytosolic loop of BOR1. A yeast two-hybrid assay supported the interaction of the AP2 complex µ-subunit with the C-terminal tail but not with the YxxΦ motifs in the cytosolic loop of BOR1. Intriguingly, lack of the AP2 subunit did not affect the B-induced rapid internalization/vacuolar sorting of BOR1. Consistent with defects in the polar localization, the AP2 complex mutants showed hypersensitivity to B deficiency. Our results indicate that AP2-dependent endocytosis maintains the polar localization of BOR1 to support plant growth under low-B conditions, whereas the B-induced vacuolar sorting of BOR1 is mediated through an AP2-independent endocytic pathway.

Glassin, a histidine-rich protein from the siliceous skeletal system of the marine sponge Euplectella, directs silica polycondensation.

The hexactinellids are a diverse group of predominantly deep sea sponges that synthesize elaborate fibrous skeletal systems of amorphous hydrated silica. As a representative example, members of the genus Euplectella have proved to be useful model systems for investigating structure-function relationships in these hierarchically ordered siliceous network-like composites. Despite recent advances in understanding the mechanistic origins of damage tolerance in these complex skeletal systems, the details of their synthesis have remained largely unexplored. Here, we describe a previously unidentified protein, named "glassin," the main constituent in the water-soluble fraction of the demineralized skeletal elements of Euplectella. When combined with silicic acid solutions, glassin rapidly accelerates silica polycondensation over a pH range of 6-8. Glassin is characterized by high histidine content, and cDNA sequence analysis reveals that glassin shares no significant similarity with any other known proteins. The deduced amino acid sequence reveals that glassin consists of two similar histidine-rich domains and a connecting domain. Each of the histidine-rich domains is composed of three segments: an amino-terminal histidine and aspartic acid-rich sequence, a proline-rich sequence in the middle, and a histidine and threonine-rich sequence at the carboxyl terminus. Histidine always forms HX or HHX repeats, in which most of X positions are occupied by glycine, aspartic acid, or threonine. Recombinant glassin reproduces the silica precipitation activity observed in the native proteins. The highly modular composition of glassin, composed of imidazole, acidic, and hydroxyl residues, favors silica polycondensation and provides insights into the molecular mechanisms of skeletal formation in hexactinellid sponges.

Evolutionary Divergence of Plant Borate Exporters and Critical Amino Acid Residues for the Polar Localization and Boron-Dependent Vacuolar Sorting of AtBOR1.

Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments.