Identification of microtubule binding sites in the Ncd tail domain

Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor pro tein that is required for spindle: assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos. Ncd has an ATP-i ndependent MT binding site in the N-terminal tail domain, and an ATP-depend ent MT binding site in the C-terminal motor domain. The ability of Ncd to c ross-link MTs through the action of the-se binding sites may be important f or Ncd function in vivo. To identify the region(s) responsible for ATP-inde pendenr MT interactions of Ncd, 12 cDNAs coding various regions of Ncd tail domain were expressed in E. coli as C-terminal fusions to thioredoxin (Trx ). Ncd tail fusion proteins (TrxNT) were purified by ion exchange (S-Sephar ose) and/or Talon metal affinity chromatography, Purified TrxNT and NT prot eins were analyzed in microtubule (MT) cosedimentation and bundling assays to identify which tail proteins were able to bind and bundle MTs. Based on the results of these experiments, all TrxNT and NT proteins that showed MT binding activity also bundled MTs, and there are two ATP-independent MT int eraction sites in the tail region: one within amino acids 83-100 that exhib its conformation-independent, high-affinity MT binding activity; and anothe r within amino acids 115-187 hat exhibits conformation-dependent, lower aff inity NIT binding activity. It is possible that both of these MT interactin g sites combine in the native protein to form a single MT binding site that allows the Ncd tail to bind cargo MTs in vivo.