Glycosylphosphatidylinositol (GPI) recognition by GPI transamidase

Abstract

GPI-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT), a penta-subunit membrane-bound enzyme. The enzyme recognizes a C-terminal signal sequence in the pro-protein and replaces it with a preformed GPI lipid. We developed detergent-solubilization conditions that permit detection of interactions between GPIT and its GPI and pro-protein substrates in pull-down assays. Using these procedures we showed that the polytopic Gaa1 subunit of GPIT is functionally important for recognition of GPI by the enzyme (JBC 279: 6540–6545 (2004)). We now extend these studies to establish the structural features of GPI that are required for GPI::GPIT interaction in the pull down assay. Our results indicate that GPI structures must contain a single phospho-ethanolamine (P-EtN) residue anywhere on the mannose core in order to be recognized by GPIT. Thus GPI anchor precursors such as H7 (EtN-P-6Manα1,2Manα1-6(EtN-P-2)Manα1,4GlcN-acyl PI) and even P-EtN-containing GPI molecules that cannot serve as protein anchors such as H6 (Manα1,2Manα1-6(EtN-P-2)Manα1,4GlcN-acyl PI) are pulled down by GPIT, whereas GPIs such as H4 (Manα1,2Manα1-6Manα1,4GlcN-acylPI) that lack P-EtN are not. The implications of these data will be discussed. Supported by NIH GM55427.