Gene Ontology annotation through association of InterPro records with GO terms
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Two different InterPro signatures drive the GO_REF:0000002 IEA annotations of PIGK. The dedicated GPI_transamidase signature (IPR028361) supplies GPI-anchor transamidase activity (GO:0003923), the specific and correct call. The generic peptidase C13 (legumain-like) fold signature (IPR001096) separately supplies peptidase activity (GO:0008233) and proteolysis (GO:0006508); these are fold-derived generalizations of the transamidation chemistry rather than descriptions of what PIGK does.
Annotation inferences using phylogenetic trees
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PAINT phylogenetic inference places PIGK in the GPI8/Gpi8p clade of catalytic GPI transamidase subunits, supporting the IBA annotations to GPI-anchor transamidase activity (GO:0003923), GPI-anchor transamidase complex (GO:0042765) and attachment of GPI anchor to protein (GO:0016255).
Gene Ontology annotation based on UniPathway vocabulary mapping
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UniPathway vocabulary mapping supplies the IEA annotation of PIGK to GPI anchor biosynthetic process (GO:0006506), a related, broader process term covering the pathway that culminates in the specific transamidation step (GO:0016255) that PIGK actually catalyzes. Note that GO:0006506 is not an is_a/part_of ancestor of GO:0016255 in the current ontology.
Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt
Combined Automated Annotation using Multiple IEA Methods
Gaa1p and gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins.
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Conserved cysteine and histidine residues of Gpi8p/PIGK, characteristic of the cysteine protease family, are essential for forming the carbonyl intermediate, identifying PIGK as the catalytic component that cleaves the GPI attachment signal peptide.
"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."
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Gaa1p and Gpi8p associate with one another and together constitute a GPI transamidase, the basis for the PIGK-GPAA1 protein-binding and complex-membership annotations.
"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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The reaction PIGK carries out replaces a C-terminal GPI attachment signal peptide with a preassembled GPI in the ER, proceeding through a carbonyl intermediate.
"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."
PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8.
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PIG-S and PIG-T form a complex with GAA1 and GPI8/PIGK, extending the transamidase to four known subunits and grounding the GPI-anchor transamidase complex (GO:0042765) annotation.
"We also demonstrate that PIG-S and PIG-T form a protein complex with GAA1 and GPI8, and that PIG-T maintains the complex by stabilizing the expression of GAA1 and GPI8."
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The GPI transamidase acts in the endoplasmic reticulum, replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI - the experimental basis for the EXP ER-membrane localization of PIGK.
"The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI."
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Knockout of PIG-S or PIG-T impairs transfer of GPI to proteins, particularly formation of the carbonyl intermediate, showing that the PIGK catalytic step depends on the intact complex.
"PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates."
Two subunits of glycosylphosphatidylinositol transamidase, GPI8 and PIG-T, form a functionally important intermolecular disulfide bridge.
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PIGK (GPI8) and PIG-T form an intermolecular disulfide bond between conserved cysteines that is required for full transamidase activity, a specific structural interaction within the complex rather than a generic protein-binding observation.
"Here we report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T, form a functionally important disulfide bond between conserved cysteine residues."
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Cysteine-to-serine mutants of GPI8 or PIG-T fail to fully restore GPI-anchored protein surface expression and have markedly reduced in vitro transamidase activity, so the bond is required for full but not minimal activity.
"Microsomal membranes of these transfectants had markedly decreased activities in an in vitro transamidase assay. The formation of this disulfide bond is not essential but required for full transamidase activity."
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A catalytically inactive human transamidase complex containing non-functional GPI8 co-purifies with proform substrate proteins, showing the five-subunit complex is sufficient to bind substrate independently of catalysis by PIGK.
"We also demonstrate that an inactive human GPI transamidase complex that consists of non-functional GPI8 and four other components was co-purified with the proform of substrate proteins, indicating that these five components are sufficient to hold the substrate proteins."
Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins.
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PIG-U was identified as the fifth subunit by affinity purification of the transamidase complex through epitope-tagged GPI8/PIGK, direct evidence for the composition of the PIGK-containing complex.
"The GPI transamidase complex affinity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U and four other known components."
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Cells lacking PIG-U still assemble the other four subunits but cannot cleave the GPI attachment signal peptide, showing that PIGK's catalytic step requires the full pentamer rather than the PIGK-GAA1 core alone.
"Cells lacking PIG-U formed complexes of the four other components normally but had no ability to cleave the GPI attachment signal peptide."
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PIG-U is proposed to recognize the GPI attachment signal or the lipid portion of GPI, dividing substrate recognition between subunits rather than assigning it all to the catalytic PIGK.
"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."
Defining the membrane proteome of NK cells.
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PIGK was detected in a high-throughput membrane-proteome survey of the NK-like YTS cell line; the study assigns no function and supports only the generic membrane (GO:0016020) annotation, not the specific ER-membrane localization.
"Mass spectrometric analysis identified 1843 proteins with high confidence scores. On the basis of the presence of transmembrane regions or evidence of posttranslational modifications and prediction algorithms, approximately 40% of the identified proteins were predicted as plausible membrane proteins."
Architecture of the human interactome defines protein communities and disease networks.
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The PIGK interaction sourced from this paper comes from BioPlex 2.0, an affinity-purification mass-spectrometry network of over 56,000 candidate interactions; it is a discovery-scale resource rather than PIGK-focused evidence.
"Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome"
Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.
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The PIGK interaction sourced from this paper comes from BioPlex 3.0 and a parallel HCT116 network, again a proteome-scale AP-MS resource rather than a PIGK-specific experiment.
"The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins."
Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit.
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GPI-TA is a five-subunit complex (PIGK, GPAA1, PIGT, PIGS, PIGU) in which loss of any one subunit abolishes activity, the clearest statement that PIGK's catalytic activity is only realized in the assembled complex.
"GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity."
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The complex recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins and then transfers GPI to the newly exposed C-terminus, the two-step reaction PIGK catalyzes.
"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."
Structure of human glycosylphosphatidylinositol transamidase.
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The 3.1 A cryo-EM structure of the human GPI transamidase identifies PIGK as the catalytic subunit and resolves a C206-H164-N58 triad critical for the transamination reaction.
"The PIGK subunit functions as the catalytic component, in which we identified a C206-H164-N58 triad that is critical for the transamination reaction."
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Transmembrane helices of the complex form a widely opened cleft directly underneath PIGK that serves as the GPI substrate-binding site, indicating that the lipid-binding pocket is a property of the assembled complex rather than of the PIGK catalytic domain alone.
"Transmembrane helices constitute a widely opened cleft, which is located underneath PIGK, serving as a GPI substrate-binding site."
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The ubiquitin E3 ligase RNF121 is resolved bound at the back of the complex and is proposed to act as a quality-control factor for the transamidase.
"The ubiquitin E3 ligase RNF121 is visualized at the back of the complex and probably serves as a quality control factor for the GPIT complex."
Molecular insights into biogenesis of glycosylphosphatidylinositol anchor proteins.
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A 2.53 A cryo-EM structure shows the human GPI transamidase is an equimolar heteropentamer, the structural basis for PIGK's part_of annotation to the GPI-anchor transamidase complex.
"Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GPI-T at a global 2.53-Å resolution, revealing an equimolar heteropentameric assembly."
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Structure-based mutagenesis supports a legumain-like mechanism for recognition and cleavage of the proprotein substrate, consistent with PIGK's peptidase C13 family assignment being mechanistically real rather than merely a fold resemblance.
"Structure-based mutagenesis suggests a legumain-like mechanism for the recognition and cleavage of proprotein substrates, and an endogenous GPI in the structure defines a composite cavity for the lipid substrate."
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The active site is an elongated composite cavity extending about 22 A from the membrane to the catalytic dyad, shaped to accommodate both the proprotein and the lipid substrate - a complex-level architecture rather than a PIGK-only active site.
"This elongated active site, stemming from the membrane and spanning an additional ~22-Å space toward the catalytic dyad, is structurally suited for both substrates which feature an amphipathic pattern that matches this geometry."
Structures of liganded glycosylphosphatidylinositol transamidase illuminate GPI-AP biogenesis.
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Substrate- and product-bound human GPI-T structures explain how the enzyme achieves broad proprotein specificity despite the absence of a consensus signal sequence, and define the catalytic mechanism of the PIGK subunit.
"Here, substrates- and products-bound human GPI-T structures identify subsite features that enable broad proprotein specificity, inform catalytic mechanism, and reveal a multilevel safeguard mechanism against its promiscuity."
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In the resting enzyme the catalytic site is occluded by a locally stabilized autoinhibitory loop, and activation requires energetically unfavourable rearrangements that convert that loop into catalytic cleft elements - a safeguard against indiscriminate cleavage.
"In the absence of proproteins, the catalytic site is invaded by a locally stabilized loop. Activation requires energetically unfavorable rearrangements that transform the autoinhibitory loop into crucial catalytic cleft elements."
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Substrate binding in the transmembrane and lumenal domains respectively powers the conformational rearrangement and induces a competent cleft, so specificity is an emergent property of the whole complex rather than of the PIGK active site alone.
"Enzyme-proprotein binding in the transmembrane and luminal domains respectively powers the conformational rearrangement and induces a competent cleft."
Multimodal cell maps as a foundation for structural and functional genomics.
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The PIGK interaction sourced from this paper comes from a multimodal subcellular map of over 5,100 proteins in U2OS cells built from combined interaction and immunofluorescence data; it is a resource-scale assignment, not a PIGK-specific assay.
"Here we construct a global map of human subcellular architecture through joint measurement of biophysical interactions and immunofluorescence images for over 5,100 proteins in U2OS osteosarcoma cells."
The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase.
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The human class K GPI-anchoring mutant, which accumulates assembled GPI donors but cannot transfer them to proproteins, is defective in the human homolog of yeast GPI8 (PIGK), identifying PIGK as the gene underlying the transamidation defect.
"we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer."
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Reconstitution of class K cells with hGPI8/PIGK abolishes GPI precursor accumulation and restores C-terminal processing of GPI-anchored proteins, the IDA basis for the attachment of GPI anchor to protein (GO:0016255) annotation.
"Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins."
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hGPI8/PIGK also restores the ability of mutant microsomes to form the active carbonyl intermediate with a proprotein, direct evidence that PIGK acts at the catalytic step of transamidation.
"hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase."
Attachment of GPI anchor to uPAR
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Reactome models the PIGK-containing transamidase complex attacking nascent uPAR in the ER lumen, cleaving after residue 305 and replacing the released C-terminal peptide with an acylated GPI moiety - a worked substrate-level instance of the GO:0016255 process.
"As nascent uPAR polypeptide moves into the lumen of the endoplasmic reticulum, it is attacked by a transamidase complex that cleaves the uPAR polypeptide after residue 305, releasing the carboxyterminal peptide of uPAR and replacing it with an acylated GPI moiety."
uPAR precursor + acyl-GPI -> uPAR-acyl-GPI + uPAR propeptide
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The reaction record attributes catalysis to GPI transamidase, a complex of at least five proteins on the lumenal surface of the ER membrane, supporting both the transamidase-activity and ER-membrane TAS annotations for PIGK.
"The reaction is catalyzed by GPI transamidase, a complex of at least five proteins associated with the lumenal surface of the endoplasmic reticulum membrane"
UniProtKB entry Q92643 (GPI8_HUMAN)
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UniProt records PIGK as the catalytic subunit of the GPI-anchor transamidase complex that forms the linkage between a proprotein and a GPI-anchor.
"FUNCTION: Catalytic subunit of the glycosylphosphatidylinositol-anchor"
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UniProt describes PIGK's substrate recognition as a C-terminal signal peptide region lacking a consensus sequence, explaining why the enzyme needs the multilevel specificity safeguards seen structurally.
"Recognizes diverse proproteins at a"
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UniProt places PIGK in a heteropentamer with PIGT, PIGS, PIGU and GAA1, and records the PIGT disulfide link that stabilizes GAA1 and PIGK.
"SUBUNIT: Heteropentamer (PubMed:35551457). Part of the GPI-anchor"
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UniProt gives PIGK's location as the endoplasmic reticulum membrane with a single-pass type I topology.
"SUBCELLULAR LOCATION: Endoplasmic reticulum membrane"