Palmitoylation by PAZ2 of Dictyostellium discoidium

The interesting life cycle of D. discoidium involves single cell growth by standard mitosis while following a period of general population starvation, cells enter a developmental phase to their life cycle. In the developmental phase cells will aggregate into a multicellular organism and begin a process of transformation involving numerous changes in gene expression. Cell-cell communication via plasma membrane receptor plays a crucial role in this process. We are studying a family of enzyme that modify proteins with palmitic acid which localizes them to the plasma membrane. This gene family named “PAZ” in Dictyostellium are protein acyltransferases. WE are analyzing the function of the paz2 gene, through the generation of knock-in and knock-out mutations. The knock-out construct was formed through a TOPO-Blunt vector containing PAZ2 which was cut with ClaI and ligated with a BSR encoding gene. The knock-in construct consisted of an eYFP-BSR tag following a NotI restriction site point mutagenesis replacing the stop codon of the PAZ2 gene. D. discoidium cells will be transformed through electroporation and observed for any abnormal phenotype for the knock-out cell line. For the knock-in which adds the fluorescent protein YFP to the protein, we will be able to visualize the expression and localization of PAZ2.

Faculty Mentor: Robert Gundersen