scholarly journals Function of AtPGAP1 in GPI anchor lipid remodeling and transport to the cell surface of GPI-anchored proteins

2021 ◽  
Author(s):  
César Bernat-Silvestre ◽  
Judit Sanchez-Simarro ◽  
Yingxuan Ma ◽  
Kim Johnson ◽  
Fernando Aniento ◽  
...  
Keyword(s):  
Cell Surface ◽  
Acyl Chain ◽  
Growth Stress ◽  
Loss Of Function ◽  
Gpi Anchor ◽  
C Terminus ◽  
Gpi Transamidase ◽  
Er Export ◽  
Inositol Ring

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.

Abstract 52: The Role of Kindlin-2 in Integrin Activation Depends on a Short C-Terminal Region

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Jamila Hirbawi ◽  
Kamila Bledzka ◽  
Yan Qing Ma ◽  
Jun Qin ◽  
Edward F Plow
Keyword(s):  
Amino Acids ◽  
Point Mutation ◽  
Single Point ◽  
Terminal Region ◽  
Membrane Receptors ◽  
Single Point Mutation ◽  
Loss Of Function ◽  
Integrin Activation ◽  
C Terminus

Integrins are heterodimeric cell membrane receptors that regulate cell adhesion, migration, and survival. The kindlins are known to be key regulators of integrin activation, the transition from a low affinity, default state to a high affinity state for ligand. This function depends on their binding, together with talin, to the cytoplasmic tails (CT) of the β subunit of integrins. Kindlins are FERM domain containing proteins, and it is its F3 (PTB) subdomain of the FERM that is the primary binding site for integrin β CT. At its very C-terminus, beyond the F3, is a short extension of 21 amino acids, K2 660-680, and we have focused on the role of this region in the co-activator function of kindlin-2 (K2). For this analysis, we performed PAC-1 (antibody to detect activated αIIbβ3 integrin) binding assays in CHO cells stably expressing integrin α IIb β 3 that were transiently transfected with talin head domain and K2 mutants. Expression levels of all proteins were verified to be similar by western blotting and FACS. Truncation of K2 at residue 660 essentially eliminated the co-activator function of K2. Deletion of smaller segments also reduced co-activator activity by 50% to 100%. Deletion of just the last two amino acids in the sequence, W 679 V 680 , resulted in a 50% reduction in co-activator activity and a single point mutation of Y 673 A also led to a 50% loss of function. A combination mutant consisting of the W 679 V 680 deletion and the Y 673 point mutation resulted in 100% loss of kindlin-2 co-activator activity. Pull-down experiments performed using GST tagged β 3 CT and CHO lysates transfected with GFP-kindlin-2 forms suggested that the C-terminal deletion did not disrupt binding to β 3 CT. This observation was corroborated by surface plasmon resonance studies in which the binding of full-length K2 and K2Δ666C (Δ666) was compared, and their K D values for immobilized β3 CT were found to be essentially the same. Overall, these data establish an important and unanticipated role of the carboxy-terminal region of kindlin-2 in its integrin co-activator function that is not dependent of its binding to integrin.

Phospholipase Resistance of the Glycosyl-Phosphatidylinositol Membrane Anchor on Human Alkaline Phosphatase

1992 ◽  
Vol 38 (12) ◽  
pp. 2517-2525 ◽  
Author(s):  
Y W Wong ◽  
M G Low
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Surface ◽  
Human Cell Lines ◽  
Membrane Anchor ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
In Vivo Release ◽  
Mammalian Tissues ◽  
Inositol Ring

Abstract Alkaline phosphatase (ALP) is attached to the cell surface in mammalian tissues via a glycosyl-phosphatidylinositol (GPI) anchor and can be released from the membrane by GPI-specific phospholipases. In a range of cultured human cell lines, however, the sensitivity of ALP to phospholipases was observed to be variable in magnitude (approximately 20-90%). The mechanism of phospholipase resistance was explored with phospholipases of different bond specificities. The results suggest that phospholipase resistance is the result of acylation of the inositol ring in the GPI anchor. The occurrence of phospholipase-resistant forms of ALP may have important implications for the in vivo release and disposition of plasma ALP.

scholarly journals FAP-1 Association with Fas (Apo-1) Inhibits Fas Expression on the Cell Surface

2003 ◽  
Vol 23 (10) ◽  
pp. 3623-3635 ◽  
Author(s):  
Vladimir N. Ivanov ◽  
Pablo Lopez Bergami ◽  
Gabriel Maulit ◽  
Taka-Aki Sato ◽  
David Sassoon ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fas Ligand ◽  
Surface Expression ◽  
Dominant Negative ◽  
Intracellular Pool ◽  
C Terminus ◽  
Interfering Rna ◽  
Insight Into ◽  
Advanced Tumors

ABSTRACT As revealed by intracellular pools of nonactive Fas (Apo-1), export of Fas to the cell surface is often impaired in human tumors, thereby inactivating Fas ligand-mediated apoptosis. Here, we demonstrate that association with Fas-associated phosphatase 1 (FAP-1) attenuates Fas export to the cell surface. Forced expression of FAP-1 reduces cell surface Fas levels and increases the intracellular pool of Fas within the cytoskeleton network. Conversely, expression of dominant-negative forms of FAP-1, or inhibition of FAP-1 expression by short interfering RNA, efficiently up-regulates surface expression of Fas. Inhibition of Fas surface expression by FAP-1 depends on its association with the C terminus of Fas. Mutation within amino acid 275 results in decreased association with FAP-1 and greater export of Fas to the cell surface in melanomas, normal fibroblasts, or Fas null cells. Identifying the role of FAP-1 in binding to, and consequently inhibition of, Fas export to the cell surface provides novel insight into the mechanism underlying the regulation of Fas trafficking, which is commonly impaired in advanced tumors with FAP-1 overexpression.

Stabilization of the angiotensin-(1–7) receptor Mas through interaction with PSD95

2013 ◽  
Vol 453 (3) ◽  
pp. 345-356 ◽  
Author(s):  
Weihua Bian ◽  
Licui Sun ◽  
Longyan Yang ◽  
Ji-Feng Li ◽  
Jia Hu ◽  
...  
Keyword(s):  
Proteasome Inhibitor Mg132 ◽  
Loss Of Function ◽  
Mas Receptor ◽  
C Terminus ◽  
Proteolytic Pathway ◽  
Intracellular Regulation ◽  
Subsequent Degradation ◽  
Ubiquitin Proteasome ◽  
Dose Dependent

The functions and signalling mechanisms of the Ang-(1–7) [angiotensin-(1–7)] receptor Mas have been studied extensively. However, less attention has been paid to the intracellular regulation of Mas protein. In the present study, PSD95 (postsynaptic density 95), a novel binding protein of Mas receptor, was identified, and their association was characterized further. Mas specifically interacts with PDZ1-2, but not the PDZ3, domain of PSD95 via Mas-CT (Mas C-terminus), and the last four amino acids [ETVV (Glu-Thr-Val-Val)] of Mas-CT were determined to be essential for this interaction, as shown by GST pull-down, co-immunoprecipitation and confocal co-localization experiments. Gain-of-function and loss-of-function studies indicated that PSD95 enhanced Mas protein expression by increasing the stabilization of the receptor. Mas degradation was robustly inhibited by the proteasome inhibitor MG132 in time- and dose-dependent manners, and the expression of PSD95 impaired Mas ubiquitination, indicating that the PSD95–Mas association inhibits Mas receptor degradation via the ubiquitin–proteasome proteolytic pathway. These findings reveal a novel mechanism of Mas receptor regulation by which its expression is modulated at the post-translational level by ubiquitination, and clarify the role of PSD95, which binds directly to Mas, blocking the ubiquitination and subsequent degradation of the receptor via the ubiquitin–proteasome proteolytic pathway.

scholarly journals Structural determinants regulating cell surface targeting of melanocortin receptors

2013 ◽  
Vol 51 (2) ◽  
pp. R23-R32 ◽  
Author(s):  
A R Rodrigues ◽  
D Sousa ◽  
H Almeida ◽  
A M Gouveia
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Melanocortin Receptors ◽  
Control Mechanisms ◽  
Structural Determinants ◽  
Surface Integration ◽  
C Terminus ◽  
G Protein Coupled

Melanocortin receptors (MCRs) belong to the G-protein-coupled receptor family of transmembrane proteins. They recognize specific ligands named melanocortins that are mainly produced in the pituitary and hypothalamus. Newly synthesized MCRs at the endoplasmic reticulum are subjected to quality control mechanisms that screen for the correct structure, folding or processing, essential for their proper cell surface expression. Some motifs, located at the N- or C-terminus or even on transmembrane and in loop regions, have been implicated in these biological processes. This article reviews these specific domains and the role of accessory proteins and post-translation modifications in MCRs' targeting to cell surface. Additionally, promising approaches involving pharmacological stabilization of misfolded and misrouted mutant MCRs, which improve their forward transport, are reported. Understanding the MCRs' structural determinants fundamental for their proper cell surface integration is essential for correcting abnormalities found in some diseases.

Role of C-terminus of Kir7.1 potassium channel in cell-surface expression

2006 ◽  
Vol 30 (3) ◽  
pp. 270-277 ◽  
Author(s):  
Toru Tateno ◽  
Nobuhiro Nakamura ◽  
Yukio Hirata ◽  
Shigehisa Hirose
Keyword(s):  
Cell Surface ◽  
Potassium Channel ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
C Terminus

scholarly journals Gis4, a New Component of the Ion Homeostasis System in the Yeast Saccharomyces cerevisiae

2006 ◽  
Vol 5 (10) ◽  
pp. 1611-1621 ◽  
Author(s):  
Tian Ye ◽  
Raúl García-Salcedo ◽  
José Ramos ◽  
Stefan Hohmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Salt Tolerance ◽  
Cell Surface ◽  
Ion Homeostasis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Snf1 Kinase ◽  
C Terminus ◽  
Surface Localization ◽  
As Stress

ABSTRACT Gis4 is a new component of the system required for acquisition of salt tolerance in Saccharomyces cerevisiae. The gis4Δ mutant is sensitive to Na+ and Li+ ions but not to osmotic stress. Genetic evidence suggests that Gis4 mediates its function in salt tolerance, at least partly, together with the Snf1 protein kinase and in parallel with the calcineurin protein phosphatase. When exposed to salt stress, mutants lacking gis4Δ display a defect in maintaining low intracellular levels of Na+ and Li+ ions and exporting those ions from the cell. This defect is due to diminished expression of the ENA1 gene, which encodes the Na+ and Li+ export pump. The protein sequence of Gis4 is poorly conserved and does not reveal any hints to its molecular function. Gis4 is enriched at the cell surface, probably due to C-terminal farnesylation. The CAAX box at the C terminus is required for cell surface localization but does not seem to be strictly essential for the function of Gis4 in salt tolerance. Gis4 and Snf1 seem to share functions in the control of ion homeostasis and ENA1 expression but not in glucose derepression, the best known role of Snf1. Together with additional evidence that links Gis4 genetically and physically to Snf1, it appears that Gis4 may function in a pathway in which Snf1 plays a specific role in controlling ion homeostasis. Hence, it appears that the conserved Snf1 kinase plays roles in different pathways controlling nutrient as well as stress response.

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

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.

scholarly journals Role of C-Terminal Domains in Surface Attachment of the Fructosyltransferase of Streptococcus salivariusATCC 25975

1998 ◽  
Vol 180 (23) ◽  
pp. 6400-6403 ◽  
Author(s):  
Catherine Rathsam ◽  
Nicholas A. Jacques
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Streptococcus Salivarius ◽  
Surface Attachment ◽  
C Terminus ◽  
Anchoring Motif ◽  
Terminal Domains

ABSTRACT The cell-associated β-d-fructosyltransferase ofStreptococcus salivarius, which is devoid of the cell wall anchoring motif, LPXTG, is released on exposure to its substrate, sucrose. Deletions within the C terminus of the enzyme implicated both the hydrophobic and the proline-glycine-serine-threonine-rich wall-associated domain in stabilizing the enzyme on the cell surface.

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