Grant: Discovery Grants Program - Individual

Our goal is to learn more about processes regulating protein localization. Many proteins are precisely targeted to particular sub-cellular compartments by specific localization signals. Correct localization is necessary to ensure that the appropriate activity is not only at the appropriate site within the cell, but also to restrict function at inappropriate sites. Nuclear import of proteins is typically mediated by interaction with soluble cytosolic receptor proteins via nuclear localization signals (NLSs). Canonical NLSs contain*either a short stretch of basic amino acids (monopartite) or two closely spaced short stretches of basic amino acids (bipartite). Despite the*short length of canonical NLSs, which are as little as 4 or 5 amino acids in length, they make high affinity interactions in the cytosol with*members of the importin family of NLS receptors (also known as karyopherins). Importins function as carriers to direct the interacting cargo to the nucleus.*It has become increasingly apparent that the many nuclear proteins do not utilize the canonical import pathway or interact with the canonical import pathway via alternative means. Indeed, ""proteome wide"" analyses in murine and yeast models found that 40-60%*of nuclear proteins do not contain a recognizable NLS. Some of these proteins may access the nucleus by other means, while other may use the conventional import apparatus in an alternative manner. As such, identification and studies of non-conventional NLSs and their mechanisms of nuclear*import remains an important area of investigation in the field of cell biology.*We adopted and improved a transcription based nuclear import assay in yeast to identify and characterize novel NLSs. This system is somewhat akin to a yeast 1-hybrid system, in that fusion of a functional NLS to a chimeric transcription factor*induces nuclear localization, allowing activation of easily detected reporter genes. This is a robust, quantitative*and well characterized system that we and others have used to map and characterize novel NLSs. The advantage of this yeast genetic approach*is that, like the yeast 2-hybrid*system, we can readily scale it up to examine massive numbers of short peptide sequences for those that confer*nuclear localization. We are doing this using a recombination system to screen a large library of short random peptides for novel linear*sequences that induce nuclear localization in the yeast nuclear import assay.*The targets of these interaction motifs will also be identified.*Our studies will identify short linear amino acid sequences that confer nuclear*localization. Characterization of these signals will identify novel mechanisms utilized to gain passage to the nucleus. As these signals function as protein interaction motifs, we will also be expanding the known repertoire of linear motifs and their targets, which play key roles in signalling and regulatory networks.*