Abstract: Glycosylphosphatidylinositol (GPI)-anchored cell wall proteins (GPI-CWPs) play an important role in the structure and function of the cell wall in Saccharomyces cerevisiae and other fungi. While the majority of characterized fungal GPI-anchored proteins localize to the cell wall, a subset of GPI proteins are thought to reside at the plasma membrane and not to traffic significantly to the cell wall. The amino acids immediately upstream of the site of GPI anchor addition (the omega site) are the primary signal determining whether a GPI protein localizes to the cell wall or to the plasma membrane. Here, evidence was found that in addition to this omega-proximal signal, other sequences in the protein can impact the distribution of GPI proteins between cell wall and membrane. In particular, it was found that long regions rich in serine and threonine residues (a feature of many cell wall proteins) can override the omega-proximal signal and redirect a model GPI plasma membrane protein to the cell wall.