Abstract
Glycosylphosphatidylinositol (GPI) transamidase (GPIT), the enzyme that attaches GPI anchors to proteins as they enter the lumen of the endoplasmic reticulum, is a membrane-bound hetero-pentameric complex consisting of Gpi8, Gpi16, Gaa1, Gpi17 and Gab1. Here, we expressed and purified the luminal domain of Saccharomyces cerevisiae (S. cerevisiae) Gpi8 using different expression systems, and examined its interaction with insect cell expressed luminal domain of S. cerevisiae Gpi16. We found that the N-terminal caspase-like domain of Gpi8 forms a disulfide-linked dimer, which is strengthened by N-glycosylation. The non-core domain of Gpi8 following the caspase-like domain inhibits this dimerization. In contrast to the previously reported disulfide linkage between Gpi8 and Gpi16 in human and trypanosome GPIT, our data show that the luminal domains of S. cerevisiae Gpi8 and S. cerevisiae Gpi16 do not interact directly, nor do they form a disulfide bond in the intact S. cerevisiae GPIT. Our data suggest that subunit interactions within the GPIT complex from different species may vary, a feature that should be taken into account in future structural and functional studies.
Highlights
Suggested to recognize long chain fatty acids of the GPI lipid mainly because its sequence contains a motif found in mammalian and yeast fatty acid elongases[5,12,18]
Towards reconstituting a GPI-transamidase (GPIT) complex for structural studies, we first investigated the interaction among the five GPIT subunits under physiological conditions
A triple tandem FLAG (3xFLAG) tag was individually engineered into the C-terminus of 4 subunits of the endogenous GPIT proteins to create 4 different strains of Saccharomyces cerevisiae (S. cerevisiae)
Summary
Suggested to recognize long chain fatty acids of the GPI lipid mainly because its sequence contains a motif found in mammalian and yeast fatty acid elongases[5,12,18]. Even though each of the GPIT subunits is postulated to have a unique function in the transamidation reaction, experimental tests of these assignments are hampered by the lack of structural information and a robust in vitro GPIT activity assay. While working towards reconstituting the S. cerevisiae GPIT complex for structural studies we made unexpected findings on the properties of S. cerevisiae Gpi[8] and S. cerevisiae Gpi[16] with regards to disulfide pairings and N-glycosylation in the Gpi8-Gpi[16] binary complex. We present these findings and discuss their implications for future structural studies on the complex
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
