Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit.

Si-Si Liu,Morihisa Fujita,Xiao-Dong Gao,Motoyuki Hattori,Yoshiko Murakami,Takayuki Kato,Yukihiko Sugita,Yi-Shi Liu,Taroh Kinoshita,Fei Jin

doi:10.3390/molecules26185462

Abstract

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Highlights

Protein anchoring by glycosylphosphatidylinositol (GPI) is a conserved posttranslational modification in eukaryotes [1,2,3,4]
Once the proteins are translocated into the luminal side of the endoplasmic reticulum (ER), the N-terminal signal sequence is cleaved by signal peptidases, and the C-terminal GPI attachment signal peptide is recognized by GPI-transamidase (GPI-TA), which transfers a GPI to the proteins [10]
PIGK is the catalytic subunit of GPI-TA, and is homologous with members of the cysteine protease family with transaminase activity [11]

Summary

Introduction

Protein anchoring by glycosylphosphatidylinositol (GPI) is a conserved posttranslational modification in eukaryotes [1,2,3,4]. GPI-APs are associated with lipid rafts and dynamic membrane domains composed of sphingolipids and cholesterol [5]. GPI biosynthesis and transfer to proteins are carried out on the endoplasmic reticulum (ER) membrane. GPI precursor proteins, which have an N-terminal signal sequence for ER translocation and a GPI attachment signal peptide at the C-terminus, are separately synthesized (Figure 1A). Once the proteins are translocated into the luminal side of the ER, the N-terminal signal sequence is cleaved by signal peptidases, and the C-terminal GPI attachment signal peptide is recognized by GPI-transamidase (GPI-TA), which transfers a GPI to the proteins [10]

Methods

Results

Conclusion