Analyzing the function of putative Mex67 phosphorylation sites in S. cerevisiae

Export of mRNA from the nucleus is essential to the viability of any eukaryotic cell. In S. cerevisiae the protein Mex67-Mtr2 plays a central role in this process as it ferries mature transcripts through the nuclear pore complex (NPC) from the nucleus to the cytoplasm. There is evidence of putative phosphorylation sites on Mex67 at residues S129, S142, and Y261. We hypothesize that phosphorylation of Mex67 regulates its function. In order to test whether phosphorylation impacts Mex67 function, the sequence encoding these amino acids were mutated to encode phospho-mimetic (S129D, S143D, and Y261E), or phospho-dead (S129A, S143A, and Y261F) residues, along with two double mutants (S129D S143D and S129A S143A). PCR mutagenesis was used to obtain seven out of eight mutants. The plasmids were then transformed into a Mex67 shuffle strain and grown on 5-FOA to assess whether the mutations were viable. We observed that all mutants were viable with no obvious growth defects at room temperature. We are currently assessing whether mutants have a temperature- sensitive growth defect. Additionally, we have cloned these mutants into yeast two-hybrid vectors to assess whether phosphorylation influences the affinity for known binding partners Nab2, NPC FG Nups, or Mtr2. Our current results suggest no functional consequence of Mex67 phosphorylation, indicating that our hypothesis may be incorrect, and updated results will be presented at the conference