Characterization of GPIT-1 and GPIT-2, two auxiliary components of the Neurospora crassa GPI transamidase complex.
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.
Bowman
Shaun M
SM
Department of Biological Sciences, University at Buffalo, Buffalo, New York 14260, USA.
Piwowar
Amy
A
Arnone
Eric D
ED
Matsumoto
Reiko
R
Koudelka
Gerald B
GB
Free
Stephen J
SJ
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006801:Humans; D007294:Inositol / Q000378:metabolism; D008968:Molecular Conformation; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D017354:Point Mutation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D005656:Fungal Proteins; D021122:Protein Subunits; D007294:Inositol; D000217:Acyltransferases; C081007:COOH-terminal signal transamidase