Structural Requirements for the Recruitment of Gaa1 into a Functional Glycosylphosphatidylinositol Transamidase Complex

Saulius Vainauskas,Yusuke Maeda,Henry Kurniawan,Taroh Kinoshita,Anant K Menon

doi:10.1074/jbc.m205402200

Abstract

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized on membrane-bound ribosomes, translocated across the endoplasmic reticulum membrane, and GPI-anchored by GPI transamidase (GPIT). GPIT is a minimally heterotetrameric membrane protein complex composed of Gaa1, Gpi8, PIG-S and PIG-T. We describe structure-function analyses of Gaa1, the most hydrophobic of the GPIT subunits, with the aim of assigning a functional role to the different sequence domains of the protein. We generated epitope-tagged Gaa1 mutants and analyzed their membrane topology, subcellular distribution, complex-forming capability, and ability to restore GPIT activity in Gaa1-deficient cells. We show that (i) detergent-extracted, Gaa1-containing GPIT complexes sediment unexpectedly rapidly at approximately 17 S, (ii) Gaa1 is an endoplasmic reticulum-localized membrane glycoprotein with a cytoplasmically oriented N terminus and a lumenally oriented C terminus, (iii) elimination of C-terminal transmembrane segments allows Gaa1 to interact with other GPIT subunits but renders the resulting GPIT complex nonfunctional, (iv) interaction between Gaa1 and other GPIT subunits occurs via the large lumenal domain of Gaa1 located between the first and second transmembrane segments, and (v) the cytoplasmic N terminus of Gaa1 is not required for formation of a functional GPIT complex but may act as a membrane-sorting determinant directing Gaa1 and associated GPIT subunits to an endoplasmic reticulum membrane domain.

Highlights

Glycosylphosphatidylinositol (GPI)1-anchored proteins are synthesized as prepro-proteins with an endoplasmic reticulum (ER)-targeting N-terminal signal sequence and a C-terminal signal sequence that directs the attachment of the GPI anchor [1,2,3,4,5]
We describe structure-function analyses of Gaa1, the most hydrophobic of the GPI transamidase (GPIT) subunits, with the aim of assigning a functional role to the different sequence domains of the protein
We show that (i) detergent-extracted, Gaa1-containing GPIT complexes sediment unexpectedly rapidly at ϳ17 S, (ii) Gaa1 is an endoplasmic reticulum-localized membrane glycoprotein with a cytoplasmically oriented N terminus and a lumenally oriented C terminus, (iii) elimination of C-terminal transmembrane segments allows Gaa1 to interact with other GPIT subunits but renders the resulting GPIT complex nonfunctional, (iv) interaction between Gaa1 and other GPIT subunits occurs via the large lumenal domain of Gaa1 located between the first and second transmembrane segments, and (v) the cytoplasmic N terminus of Gaa1 is not required for formation of a functional GPIT complex but may act as a membrane-sorting determinant directing Gaa1 and associated GPIT subunits to an endoplasmic reticulum membrane domain

Summary

Introduction

Glycosylphosphatidylinositol (GPI)1-anchored proteins are synthesized as prepro-proteins with an endoplasmic reticulum (ER)-targeting N-terminal signal sequence and a C-terminal signal sequence that directs the attachment of the GPI anchor [1,2,3,4,5]. Gaa1 Variants Containing an Intact Lumenal Loop but Lacking All Except the First Two TM Domains Are Able to Form a Complex with Gpi8, PIG-S, and PIG-T—To identify structural features of Gaa1 required for its interaction with the other known components of GPIT, we created a series of N-terminally FLAG-tagged truncation mutants (Fig. 3A) in which we systematically removed six of the seven predicted membranespanning segments from the C terminus of Gaa1.

Results

Conclusion