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

ABSTRACTGPI-anchored proteins (GPI-APs) play an important role in a variety of plant biological processes including growth, stress response, morphogenesis, signalling and cell wall biosynthesis. The GPI-anchor contains a lipid-linked glycan backbone that is synthesized in the endoplasmic reticulum (ER) where it is subsequently transferred to the C-terminus of proteins containing a GPI signal peptide by a GPI transamidase. Once the GPI anchor is attached to the protein, the glycan and lipid moieties are remodelled. In mammals and yeast, this remodelling is required for GPI-APs to be included in Coat Protein II (COPII) coated vesicles for their ER export and subsequent transport to the cell surface. The first reaction of lipid remodelling is the removal of the acyl chain from the inositol group by Bst1p (yeast) and PGAP1 (mammals). In this work, we have used a loss-of-function approach to study the role of PGAP1/Bst1 like genes in plants. We have found that Arabidopsis PGAP1 localizes to the ER and probably functions as the GPI inositol-deacylase which cleaves the acyl chain from the inositol ring of the GPI anchor. In addition, we show that PGAP1 function is required for efficient ER export and transport to the cell surface of GPI-APs.One sentence summaryGPI anchor lipid remodeling in GPI-anchored proteins is required for their transport to the cell surface in Arabidopsis.

Download Full-text

AtPIG-S, a predicted Glycosylphosphatidylinositol Transamidase Subunit, is critical for pollen tube growth in Arabidopsis

10.1101/2020.05.06.066910 ◽

2020 ◽

Author(s):

Nick Desnoyer ◽

Greg Howard ◽

Emma Jong ◽

Ravishankar Palanivelu

Keyword(s):

Pollen Tube ◽

Male Fertility ◽

Male Gametophyte ◽

Pollen Grains ◽

Unexpected Finding ◽

Gpi Anchor ◽

Developmental Defects ◽

Post Translational Modifications ◽

Gpi Transamidase

AbstractBackgroundGlycosylphosphatidylinositol (GPI) addition is one of the several post-translational modifications to proteins that increase their affinity for membranes. In eukaryotes, the GPI transamidase complex (GPI-T) catalyzes the attachment of pre-assembled GPI anchors to GPI-anchored proteins (GAPs) through a transamidation reaction. A mutation in AtGPI8 (gpi8-2), the putative catalytic subunit of GPI-T in Arabidopsis, is transmitted normally through the female gametophyte (FG), indicating the FG tolerates loss of GPI transamidation. In contrast, gpi8-2 almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that gpi8-2 FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized.ResultsHere we investigated the effect of loss of AtPIG-S, another GPI-T subunit, in both gametophytes. Like gpi8-2, we showed that a mutation in AtPIG-S (pigs-1) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG, yet is transmitted normally through the FG. Conversely, pigs-1 severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A pPIGS:PIGS-GFP transgene complemented these MG defects and enabled generation of pigs-1/pigs-1 seedlings, but seemingly failed to rescue the function of AtPIG-S in the sporophyte, as pigs-1/pigs-1, pPIGS:PIGS-GFP seedlings died soon after germination.ConclusionsCharacterization of pigs-1 provided further evidence that the FG tolerates loss of GPI transamidation more than the MG and that the MG compared to the FG may be a better haploid system to study the role of GPI-anchoring. pigs-1 pollen develops normally and thus represent a tool in which GPI anchor biosynthesis and transamidation of GAPs have been uncoupled, offering a potential way to study free GPI in plant development. While previously reported male fertility defects of GPI biosynthesis mutants could have been due either to loss of GPI or GAPs lacking the GPI anchor, our results clarified that the loss of mature GAPs underlie male fertility defects of GPI-deficient pollen grains, as pigs-1 is defective only in the downstream transamidation step. Our study also provided further evidence that GPI transamidation is essential in seedling development.

Download Full-text

Release of Alkaline Phosphatase Caused by PIGV Mutations In Patients with Hyperphosphatasia-Mental Retardation Syndrome (HPMR), a Recently Found Second Inherited GPI Anchor Deficiency.

Blood ◽

10.1182/blood.v116.21.2031.2031 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2031-2031

Author(s):

Yoshiko Murakami ◽

Peter M Krawitz ◽

Peter N Robinson ◽

Stefan Mundlos ◽

Yusuke Maeda ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Mental Retardation ◽

Signal Peptide ◽

Cho Cells ◽

Conflicts Of Interest ◽

Serum Alkaline Phosphatase ◽

Elevated Serum ◽

Common Symptom ◽

Gpi Anchor ◽

Gpi Transamidase

Abstract Abstract 2031 Recent whole exome sequencing of three sibs of non-consanguineous parents demonstrated PIGV mutations in Hyperphosphatasia-Mental Retardation syndrome (HPMR), an autosomal recessive disease characterized by mental retardation and elevated serum alkaline phosphatase (ALP) levels (Eur J Med Genet, vol.53, issue2, p85, 2010). PIGV is the second mannosyltransferase essential for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations found in four families caused amino acid substitutions A341E, A341V, Q256K and H385P. We have shown that these mutant PIGV proteins are unstable and the mutant cDNAs restored only subnormal GPI biosynthetic activity after transfection into PIGV deficient CHO cells. The backbone of GPI-anchor is synthesized in the endoplasmic reticulum (ER) and is transferred to the proteins which have GPI attachment signal at the C-terminal. It is known that GPI-anchored proteins are not expressed on the surface of GPI deficient cells due to degradation within the cells or secretion. ALP, a GPI anchored protein, was efficiently secreted into medium from PIGV deficient CHO cells, in which incomplete GPI bearing one mannose was accumulated. In contrast, ALP was degraded in PIGL, DPM2 or PIGX deficient CHO cells, in which GPIs lacking mannose were accumulated. Secretion of ALP required GPI transamidase that cleaves the C-terminal GPI attachment signal peptide and replaces it with GPI. It seems that GPI transamidase is activated by GPI bearing at least one mannose, cleaving the hydrophobic signal peptide and resulting in secretion of soluble ALP. It is well known that hypophosphatasia is caused by a deficiency of liver-, bone-, kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. This inherited disorder is characterized by defective bone mineralization and some patients develop epilepsy caused by the disturbance of pyridoxal-5-phosphate (PLP) dependent metabolism of neurotransmitters due to the defect in ALPL which converts PLP to pyridoxal (PL) thereby facilitating passive uptake of this freely diffusible form at the cell surface. Some of the HPMR patients have seizures and the patients with previously reported inherited GPI deficiency caused by defective PIGM encoding the first mannosyltransferase (Nature Med. 12, p846 2006) also have seizures. This common symptom may be caused by the loss of membrane bound ALPL. On the other hand, patients with HPMR do not have the portal vein thrombosis seen in the patients with PIGM deficiency. The affected cell types and the degree of deficiencies in various GPI-anchored proteins might be the cause of the variability of the clinical features among GPI deficiencies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

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

Molecules ◽

10.3390/molecules26185462 ◽

2021 ◽

Vol 26 (18) ◽

pp. 5462

Author(s):

Si-Si Liu ◽

Fei Jin ◽

Yi-Shi Liu ◽

Yoshiko Murakami ◽

Yukihiko Sugita ◽

...

Keyword(s):

Functional Analysis ◽

Posttranslational Modification ◽

Homologous Proteins ◽

Gpi Anchor ◽

Purification Method ◽

C Terminus ◽

Precursor Proteins ◽

Amino Acid Mutations ◽

Gpi Transamidase ◽

Particle Images

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.

Download Full-text