Hrd1-dependent degradation of the unassembled PIGK subunit of the GPI transamidase complex

Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of β-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins. While mutagenesis of auxiliary genes of this pathway suggested roles of GPI-anchored proteins in hyphal growth and virulence, essential genes of this pathway have not been characterized. Taking advantage of RNA interference (RNAi) we analyzed the function of the three essential genes GPI12, GAA1 and GPI8, encoding a cytoplasmic N-acetylglucosaminylphosphatidylinositol deacetylase, a metallo-peptide-synthetase and a cystein protease, the latter two representing catalytic components of the GPI transamidase complex. RNAi strains showed drastic cell-wall defects, resulting in exploding infection cells on the plant surface and severe distortion of in planta–differentiated infection hyphae, including formation of intrahyphal hyphae. Reduction of transcript abundance of the genes analyzed resulted in nonpathogenicity. We show here for the first time that the GPI synthesis genes GPI12, GAA1, and GPI8 are indispensable for vegetative development and pathogenicity of the causal agent of maize anthracnose, Colletotrichum graminicola.

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Truncation of the caspase-related subunit (Gpi8p) of Saccharomyces cerevisiae GPI transamidase: Dimerization revealed

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2007.03.035 ◽

2007 ◽

Vol 462 (1) ◽

pp. 83-93 ◽

Cited By ~ 8

Author(s):

Jennifer L. Meitzler ◽

Jeffrey J. Gray ◽

Tamara L. Hendrickson

Keyword(s):

Saccharomyces Cerevisiae ◽

Gpi Transamidase

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GAA1 recognizes the GPI anchor attachment signals and is complexed with GP18 to form a GPI transamidase

Molecular Immunology ◽

10.1016/s0161-5890(98)90764-7 ◽

1998 ◽

Vol 35 (6-7) ◽

pp. 388 ◽

Cited By ~ 1

Author(s):

K. Ohishi ◽

N. Inoue ◽

J. Takeda ◽

D. Hamburger ◽

H. Riezman ◽

...

Keyword(s):

Gpi Anchor ◽

Gpi Transamidase

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Human PIG-U and Yeast Cdc91p Are the Fifth Subunit of GPI Transamidase That Attaches GPI-Anchors to Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e02-12-0794 ◽

2003 ◽

Vol 14 (5) ◽

pp. 1780-1789 ◽

Cited By ~ 75

Author(s):

Yeongjin Hong ◽

Kazuhito Ohishi ◽

Ji Young Kang ◽

Satoshi Tanaka ◽

Norimitsu Inoue ◽

...

Keyword(s):

Amino Acid ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Amino Acid Identity ◽

Complementation Group ◽

Long Chain Fatty Acid ◽

Ovary Cells ◽

Eukaryotic Proteins ◽

Gpi Transamidase

Many eukaryotic proteins are anchored to the cell surface via glycosylphosphatidylinositol (GPI), which is posttranslationally attached to the carboxyl-terminus by GPI transamidase. The mammalian GPI transamidase is a complex of at least four subunits, GPI8, GAA1, PIG-S, and PIG-T. Here, we report Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the gene responsible, termed PIG-U, that encoded a 435-amino-acid hydrophobic protein. The GPI transamidase complex affinity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U and four other known components. Cells lacking PIG-U formed complexes of the four other components normally but had no ability to cleave the GPI attachment signal peptide. Saccharomyces cerevisiae Cdc91p, with 28% amino acid identity to PIG-U, partially restored GPI-anchored proteins on the surface of class U cells. PIG-U and Cdc91p have a functionally important short region with similarity to a region conserved in long-chain fatty acid elongases. Taken together, PIG-U and the yeast orthologue Cdc91p are the fifth component of GPI transamidase that may be involved in the recognition of either the GPI attachment signal or the lipid portion of GPI.

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Low-resolution structure of the soluble domain GPAA1 (yGPAA170–247) of the glycosylphosphatidylinositol transamidase subunit GPAA1 from Saccharomyces cerevisiae

Bioscience Reports ◽

10.1042/bsr20120107 ◽

2013 ◽

Vol 33 (2) ◽

Cited By ~ 4

Author(s):

Wuan Geok Saw ◽

Birgit Eisenhaber ◽

Frank Eisenhaber ◽

Gerhard Grüber

Keyword(s):

Saccharomyces Cerevisiae ◽

Solution Structure ◽

Scattering Data ◽

Radius Of Gyration ◽

Low Resolution ◽

X Ray Scattering ◽

Eukaryotic Proteins ◽

Structural Content ◽

Α Helix ◽

Gpi Transamidase

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA170–247 and yGPAA170–339 of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70–247 and 70–339 respectively. The secondary structural content of the stable and monodisperse yGPAA170–247 has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA170–247 has an Rg (radius of gyration) of 2.72±0.025 nm and Dmax (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA170–247. The large elliptical shape of yGPAA170–247 is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA170–247 will be discussed together with the recently determined low-resolution structures of yPIG-K24–337 and yPIG-S38–467 from S. cerevisiae in the GPI transamidase complex.

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Proprotein interaction with the GPI transamidase

Journal of Cellular Biochemistry ◽

10.1002/jcb.10439 ◽

2003 ◽

Vol 88 (5) ◽

pp. 1025-1037 ◽

Cited By ~ 12

Author(s):

Rui Chen ◽

Vernon Anderson ◽

Yukio Hiroi ◽

M. Edward Medof

Keyword(s):

Gpi Transamidase

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Gaa1p and Gpi8p Are Components of a Glycosylphosphatidylinositol (GPI) Transamidase That Mediates Attachment of GPI to Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.11.5.1523 ◽

2000 ◽

Vol 11 (5) ◽

pp. 1523-1533 ◽

Cited By ~ 83

Author(s):

Kazuhito Ohishi ◽

Norimitsu Inoue ◽

Yusuke Maeda ◽

Junji Takeda ◽

Howard Riezman ◽

...

Keyword(s):

Signal Peptide ◽

Posttranslational Modification ◽

Eukaryotic Cell ◽

Surface Proteins ◽

F9 Cells ◽

Precursor Proteins ◽

Key Enzyme ◽

Gpi Transamidase

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted aGAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.

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Precursors of select GPI-anchored proteins positively regulate GPI biosynthesis under the control of ERAD

10.21203/rs.3.rs-998556/v1 ◽

2021 ◽

Author(s):

Yi-Shi Liu ◽

Yicheng Wang ◽

Xiaoman Zhou ◽

LinPei Zhang ◽

Ganglong Yang ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Signal Peptide ◽

Active Element ◽

Signal Peptides ◽

Endoplasmic Reticulum Associated Degradation ◽

Genome Wide ◽

A Genome ◽

Mechanistic Basis ◽

Gpi Transamidase ◽

Regulatory Connection

Abstract We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is regulated by endoplasmic reticulum associated degradation (ERAD); however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR–Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 together upregulate GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, remaining the uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 32 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55 and CD48 enhance GPI biosynthesis. ARV1 is essential for the GPI upregulation by CD55 precursor. Our data demonstrate an ARV1-dependent regulatory connection between GPI biosynthesis and precursors of select GPI-anchored proteins that are under the control of ERAD.

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