One Single Basic Amino Acid at the ω-1 or ω-2 Site Is a Signal That Retains Glycosylphosphatidylinositol-Anchored Protein in the Plasma Membrane of Aspergillus fumigatus

Abstract

ABSTRACT Although the plasma membrane is the terminal destination for glycosylphosphatidylinositol (GPI) proteins in higher eukaryotes, cell wall-attached GPI proteins (GPI-CWPs) are found in many fungal species. In yeast, some of the cis -requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood. However, it remains to be determined how Aspergillus fumigatus , an opportunistic fungal pathogen, signals, and sorts GPI proteins to either the plasma membrane or the cell wall. In this study, chimeric green fluorescent proteins (GFPs) were constructed as fusions with putative C-terminal GPI signal sequences from A. fumigatus Mp1p, Gel1p, and Ecm33p, as well as site-directed mutations thereof. By analyzing cellular localization of chimeric GFPs using Western blotting, electron microscopy, and fluorescence microscopy, we showed that, in contrast to yeast, a single Lys residue at the ω-1 or ω-2 site alone could retain GPI-anchored GFP in the plasma membrane. Although the signal for cell wall distribution has not been identified yet, it appeared that the threonine/serine-rich region at the C-terminal half of Af Mp1 was not required for cell wall distribution. Based on our results, the cis -requirements directing localization of GPI proteins in A. fumigatus are different from those in yeast.