Abstract
Glycosylphosphatidylinositol (GPI) anchoring of proteins is catalyzed by GPI transamidase (GPIT), a multisubunit, endoplasmic reticulum (ER)-localized enzyme. GPIT recognizes ER-translocated proteins that have a GPI-directing C-terminal signal sequence and replaces this sequence with a preassembled GPI anchor. Although the GPI signal sequence has been extensively characterized, little is known about the structural features of the GPI lipid substrate that enable its recognition by GPIT. In a previous study we showed that mature GPIs could be co-immunoprecipitated with GPIT complexes containing functional subunits (Vainauskas, S., and Menon, A. K. (2004) J. Biol. Chem. 279, 6540-6545). We now use this approach, as well as a method that reconstitutes the interaction between GPIs and GPIT, to define the basis of the interaction between GPI and human GPIT. We report that (i) human GPIT can interact with GPI biosynthetic intermediates, not just mature GPIs competent for transfer to protein, (ii) the ethanolamine phosphate group on the third mannose residue of the GPI glycan is not critical for GPI recognition by GPIT, (iii) the ethanolamine phosphate residue linked to the first mannose of the GPI structure is a major feature of GPIs that is recognized by human GPIT, and (iv) the simplest GPI recognized by human GPIT is EtN-P-2Manalpha1-4GlcN-(acyl)-phosphatidyl-inositol. These studies define the molecular characteristics of GPI that are recognized by GPIT and open the way to identifying GPIT subunits that are involved in this process.
Highlights
The costs of publication of this article were defrayed in part by the payment of page charges
2 The abbreviations used are: GPI, glycosylphosphatidylinositol; ER, endoplasmic reticulum; EtN-P, ethanolamine phosphate; FLAG epitope tag, an 8-amino acid sequence consisting of DYKDDDDK; GPIT, GPI transamidase; GPI-PLD, GPI-specific phospholipase D; IP, immunoprecipitate; Jb␣m, jack bean ␣-mannosidase; ManN, mannosamine; PIG, phosphatidylinositol glycan; TM, transmembrane; V5 epitope tag, a 14-amino acid sequence consisting of GKPIPNPLLGLDST; PI, phosphatidylinositol; PNT, 1,10-phenanthroline
In an effort to shed light on this problem, we considered the following three questions. (i) Is the terminal EtN-P required for GPI interaction with GPIT? (ii) Can GPI biosynthetic intermediates bind GPIT? (iii) What is the minimal GPI structure recognized by GPIT? To answer these questions we made use of our ability to co-precipitate GPIs with epitope-tagged human GPIT complexes [36]
Summary
The costs of publication of this article were defrayed in part by the payment of page charges. To answer these questions we made use of our ability to co-precipitate GPIs (metabolically radiolabeled with [2-3H]mannose in HeLa cells) with epitope-tagged human GPIT complexes [36].
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