Grant: Discovery Grants Program - Individual

<p>Many proteins are precisely targeted to particular sub-cellular compartments by specific localization signals. 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 localization of the interacting cargo to the nucleus. The interaction of cargo/carrier complexes with components of the nuclear pore complex leads to nuclear import. </p> <p>However, it has become increasingly apparent that the many nuclear proteins do not utilize the canonical pathway or interact with the canonical pathway via alternative means. Indeed, ""genome wide"" analysis of nuclear import in the yeast <em>S. cerevisiae </em>suggests that >40% of nuclear proteins do not contain recognizable NLSs and must access the nucleus by other means. Relatively little is known about how these proteins utilize non-conventional mechanisms to access the nucleus. As such, studies of non-conventional mechanisms of nuclear import remain an important area of investigation in the field of cell biology. </p> <p><strong>Hypothesis:</strong> A high throughput transcription based nuclear import assay in yeast will identify novel short sequence motifs that confer nuclear import via non-canonical pathways or via alternative interaction strategies with canonical importin based pathways. </p> <p>My research program in nuclear import was established using a transcription based nuclear import assay in yeast that we adopted and improved. 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 of the transcription factor and the activation of easily detected reporter genes. This is a robust, quantitative and well characterized assay system that we 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 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 our yeast nuclear import assay.</p> <p><strong>Significance:</strong> Our studies will identify short linear amino acid sequences that function as protein interaction motifs to confer nuclear localization. The characterization of these non-canonical signals will identify novel mechanisms utilized to gain passage to the eukaryotic cell nucleus. </p>