Identification of tuftelin- and amelogenin-interacting proteins using the yeast two-hybrid system

Biomineralization of enamel is a complex process that involves the eventual replacement of an extracellular protein matrix by hydroxyapatite crystalli tes. To date four different enamel matrix proteins have been identified; th e amelogenins, tuftelin, enamelin and ameloblastin. Assembly of the enamel extracellular matrix from these component proteins is believed to be critic al in producing a matrix competent to undergo mineral replacement. Enamel f ormation is a complex: process and additional proteins are likely to have a role in the assembly of the extracellular matrix. In order to identify add itional proteins involved in the assembly process, the yeast two-hybrid sys tem developed by Fields and Song (1989) has been implemented. This system a llows for the identification of unknown proteins that interact with protein s of interest. Typically a known protein is used as "bait" to screen a cDNA expression library of interest. In our studies, tuftelin or amelogenin hav e been used to screen a mouse teeth library produced from one day old pups, A library screening of six million clones with amelogenin as bait resulted in eleven positive clones all of which show high homology to the human leu kocyte antigen-B (HLA-B) associated transcript (BAT) family of genes. A lib rary screening of one million clones using tuftelin as the bait identified twenty-one tuftelin-interacting proteins. Ten of these proteins are either keratin K5 or keratin K6, four are constitutively expressed and the remaini ng seven are novel. Further characterization of the proteins shown to inter act with amelogenin or tuftelin may shed additional light on this complex p rocess of enamel matrix assembly.