Solubilization and Characterization of a Membrane-Bound Auxin-Binding Protein from Cell Suspension Cultures of Nicotiana tabacum

Abstract

A membrane-bound auxin-binding protein (MABP) was solubilized by Triton X-100 from cell suspension cultures of Nicotiana tabacum L. Solubilization of MABP was dependent on the detergent concentration and more than 80% of naphthalene-l-acetic acid (NAA)-binding activity was recovered by an optimum concentration of 0.2%. The solubilized MABP was highly heat-unstable and sensitive to protease. The properties of MABP (affinity, temperature dependence, pH optimum, and analog specificity for auxin binding) did not significantly change after solubiliza-tion, e.g. the solubilized MABP showed no or very low levels of NAA-binding at 0 to 4°C but showed a high-affinity binding (dissociation constant Kd = 2.7 ± 0.3 x 10-7 M) at 25°C at an optimum pH of 5.0. NAA-binding of the solubilized MABP proceeded very slowly, i.e. a time of half-maximum binding was at least 15 minutes, although the solubi-lized MABP showed higher rates of association (k1 = 1.3 versus 0.9 x 105 M-' min-') and dissociation (k-I = 2.2 versus 1.6 x 10-2 min-') with NAA than the bound MABP. These results show that specific, saturable, and reversible auxin binding to MABP from dicotyledonous N. tabacum differs from that from monocotyledonous Zea mays, and confirm that MABP is distinct from a soluble auxin-binding protein which also is present in N. tabacum. High-affinity, auxin-binding proteins, either membrane-bound or soluble, have been described for tissues from both monoco-tyledons and dicotyledons (reviewed in Refs. 7, 17, 27). A major experimental system used for the study of auxin-binding proteins has been tissues commonly used in auxin bioassays such as excised sections of coleoptiles and stems. As an alternative auxin-responsive system, cultured plant cells and tissues have also been used because of their dependency on auxin for growth and development. Among cell and tissue culture systems Nicotiana tabacum has been unique in that the presence of both MABP2 and SABP have been demonstrated and their properties have extensively been studied (10, 14, 15, 22, 28, 29, 30). Comparisons of the properties of MABP and SABP in N. tabacum have thus far revealed significant differences between ' Supported in part by a grant-in-aid for scientific research (No. 61560005) from the Ministry ofEducation, Science and Culture ofJapan. 2Abbreviations: MABP, membrane-bound auxin-binding protein; SABP, cytoplasmic soluble auxin-binding protein