The purpose of this study is to identify proteins involved in endoplasmic reticulum to cytoplasm transport of the Gag protein of the yeast Saccharomyces cerevisiae Ty1 retroelement. Saccharomyces cerevisiae, or bakers/brewer’s yeast, exists as a model organism due to its quick growth time and easily manipulated genome. A multitude of yeast genes are also homologous to ones found in larger eukaryotes. During Ty1 transposition, Gag is translocated by the signal recognition protein (SRP) from the cytoplasm to the ER lumen where it adopts a stable conformation. The stabilized Gag protein is then translocated through an unknown mechanism to the cytoplasm where it aggregates to form virus-like particles. Screening of a yeast deletion library identified SIW14 as a potential candidate gene involved in Gag translocation. The yeast deletion library was transformed with a Ty1 Gag-Ura3 fusion protein on a LEU2 selectable plasmid. In wild type cells, the Gag-Ura3 fusion is transported to the cytoplasm, thus yeast cells are unable to survive on media containing 5-fluoroorotic acid (5-FOA), as the URA3 gene converts 5-FOA to the toxic product 5-fluorouracil. Cells with an siw14 deletion are viable on 5-FOA containing media, presumably sequestering the Ty1 Gag-Ura3 fusion protein in the ER. Siw14p is an inositol phosphatase that hydrolyzes the beta phosphate on 5-diphospho-1,2,3,4,6-pentakisphosphate (5PP-IP5) to inositol hexaphosphate (IP6). Inositol pyrophosphates are energy-rich signaling molecules and can act as biological messengers. Vip1p, Ddp1p, and Kcs1p are also proteins involved in the phosphorylation state of inositol pyrophosphates in yeast. inositol heptaphosphate (IP7) or inositol octaphosphate (IP8) are substrates for pyrophosphorylation of phosphorylated serine residues in proximity to acidic amino acids, such as aspartate and glutamate. No energy is expended during pyrophosphorylation; it is dependent on the presence of an Mg2+ divalent ion. We hypothesize that the deletion of a pyrophosphatase kinase results in the inability of an unknown transport protein from being pyrophopshoprylated to facilitate Gag transport across the ER. Strains harboring deletions of siw14, kcs1, vip1, and ddp1 will be assayed for Ty1 transposition. Fluorescence microscopy of deletion and wild type cells containing a fluorescently labeled Gag protein will be observed for Gag localization.
