Functional characterization of the Opitz syndrome gene product (midin): evidence for homodimerization and association with microtubules throughout the cell cycle

Opitz syndrome (OS) is a multiple congenital anomaly manifested by abnormal closure of midline structures. The gene responsible for the X-linked form of this disease, MID1, encodes a protein (midin) that contains a RING, two B-boxes, a coiled-coil (the so-called tripartite motif) and an RFP-like dom ain. The tripartite motif is characteristic of a family of proteins, named the B-box family, involved in cell proliferation and development. Since the subcellular compartmentalization and the ability to form multiprotein stru ctures both appear to be crucial for the function of this family of protein s; we have studied these properties on the wild-type and mutated forms of m idin, We found that endogenous midin is associated with microtubules throug hout the cell cycle, co-localizing with cytoplasmic fibres in interphase an d with the mitotic spindle and midbodies during mitosis and cytokinesis, Im munoprecipitation experiments demonstrated the ability of the tripartite mo tif to mediate midin homodimerization, consistent with the evidence, obtain ed by gel filtration analysis, that midin exists in the form of large prote in complexes, Functional characterization of altered forms of midin, result ing from mutations found in OS patients, revealed that association with mic rotubules is compromised, while the ability to homodimerize and form multip rotein complexes is retained. We suggest that midin is involved in the form ation of multiprotein structures acting as anchor points to microtubules an d that impaired association with these cytoskeletal structures causes OS de velopmental defects.