Characterization Of Pollen-Specific Microtubule-Associated Protein SB401 From Solanum Berthaultii

Microtubule associated proteins (MAPs) are the major factor involved in the regulation of microtubule (MT) dynamics and organization for functions of the MT cytoskeleton. The pollen-specific protein SB401 from Solarium berthaultii contains imperfect repeated motifs, which resembles a repetitive domain responsible for MT binding in vitro of the microtubule-associated protein MAP1B in animal cells, and also KKEE motifs in actin binding domain of villin, an F-actin binding protein to bundle actin filaments. It suggests that SB401 may bind to both MTs and actin filaments, and represents a novel pollen-specific MAP in plants.In present study, we expressed SB401 in bacterium. Purified recombinant SB401 protein was obtained to analyze the activity of SB401 protein in binding to and bundling MTs and actin filaments, and their regulatory functions in tubulin polymerization and MT dynamics. Results of co-sedimentation experiments, fluorescence and electron microscopy observations demonstrated that SB401 bound to MTs specifically, with a bind ratio of tubulin dimmer to SB401 of 1: 0.4. SB401 bundled also taxol-MTs, in a concentration dependent manner. With increase of the concentration of SB401, more MT bundles formed and cross-linked into microtubule meshworks. MTs in bundles bunched together quite tightly along their whole length, with a gap between the MTs about 6-8 nm. However, obvious filamentous cross-bridges were detected between the MTs in the bundles. SB401 proteins existed as monomers, dimmers or tetramers in the solution. All these SB401 modules could bind to MT and cause MT bundling in vitro. Our biochemical analysis also showed that SB401 bound to tubulin as well. SB401 exhibited both inhibitory or enhancing effect on tubulin polymerization, according to SB401 concentration and the temperature. At high temperature and SB401 concentrations, SB401 bound to tubulin and reduced the concentration of free tubulins, and in a consequence, inhibited tubulin polymerization. In the contrast, at low temperature and SB401 concentrations, SB401 bound preferentially to and bundled MTs, and enhanced tubulin polymerization. Double immuno- fluorescence staining also showed that SB401 was co-localized with MTs in pollen tubes.Further investigation showed that SB401 bound to actin filaments, with a binding ratio of G-actin to SB401 of 1:0.22. In addition, SB401 bundled actin filaments, too, but taking longer time to bundle actin filaments than MTs. However, SB401 had no effect on actin polymerization. SB401 bound both to and co-localized MTs and actin filaments in the presence of both MTs and actin filaments. Nevertheless, SB401 protein had a much higher affinity for MTs than actin filaments. SB401 preferentially bound MTs to form MT bundlesOur experiment results demonstrate that the SB401 protein is a novel pollen-specific MAP, which binds to and bundles both MTs and actin filaments and regulates tubulin polymerization, and therefore may have important roles in plant pollen development and pollen tube growth.