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Characterization of GPIT-1 and GPIT-2, two auxiliary components of the Neurospora crassa GPI transamidase complex

     Department of Biological Sciences, University at Buffalo, Buffalo, New York 14260

The glycosylphosphatidylinositol (GPI) transamidase contains five known subunits and functions in the lumen of the ER to produce GPI-anchored proteins. The transamidase cleaves proteins containing a GPI anchor attachment signal at their C terminus and generates an amide bond between the newly generated carboxyl terminus of the protein and a GPI anchor. We have identified and characterized GPIT-1 and GPIT-2, two of the transamidase subunits from Neurospora crassa. GPIT-1 and GPIT-2 are homologs of the human PIG-T and PIG-U transamidase subunits respectively. We demonstrated that GPIT-2 is required for the addition of GPI anchors onto GPI-anchored proteins. We employed the Neurospora RIP (repeat-induced point mutation) phenomenon to generate 106 "noncritical" amino acid changes in GPIT-1 and 84 "noncritical" amino acid changes in GPIT-2. We used the data to evaluate three-dimensional models for the structures of GPIT-1 and GPIT-2. The mutational data for GPIT-1 is consistent with a multiple-blade propeller structure containing a central channel. The mutational analysis for GPIT-2 supports a structural model based on the karyopherin alpha subunit.

Key words: Glycosylphosphatidylinositol transamidase, GPI anchor, GPI transamidase, Karyopherin alpha, PIG-T, PIG-U