How does the translocon differentiate between hydrophobic sequences that form part of either a GPI (glycosylphosphatidylinositol)-anchor signal or a stop transfer sequence?

2003 ◽  
Vol 31 (6) ◽  
pp. 1257-1259 ◽  
Author(s):  
J.A. Dalley ◽  
N.J. Bulleid
Keyword(s):  
Secretory Pathway ◽  
Lipid Membrane ◽  
Polypeptide Chain ◽  
Gpi Anchor ◽  
Major Pathway ◽  
Glycosylphosphatidylinositol Anchor ◽  
C Terminus ◽  
Lipid Moiety ◽  
Form Part ◽  
Lipid Anchor

Newly synthesized proteins entering the eukaryotic secretory pathway may be attached to the lipid membrane by essentially one of two mechanisms. They may either contain a hydrophobic stop transfer sequence that directs their integration into the bilayer with the consequence that the polypeptide spans the membrane either one or several times, or alternatively the polypeptide chain may be modified by the covalent addition of a lipid anchor resulting in the attachment of the protein to the membrane via the lipid moiety. The major pathway for the covalent addition of a lipid anchor involves the post-translational attachment of GPI (glycosylphosphatidylinositol) to the C-terminus. Proteins modified in this way contain a specific signal that is recognized by the GPI-anchor processing machinery. Hence both the integration of protein directly into the lipid bilayer and the addition of GPI anchors require the presence of sequences within the polypeptide chain to target the proteins to these pathways. This article will describe the main characteristics of these signals and their similarities and will discuss how the translocon may play a crucial role in their recognition.

scholarly journals Removal of the glycosylphosphatidylinositol anchor from PrPSc by cathepsin D does not reduce prion infectivity

2006 ◽  
Vol 395 (2) ◽  
pp. 443-448 ◽  
Author(s):  
Patrick A. Lewis ◽  
Francesca Properzi ◽  
Kanella Prodromidou ◽  
Anthony R. Clarke ◽  
John Collinge ◽  
...  
Keyword(s):  
Cathepsin D ◽  
Rocky Mountain ◽  
Brain Homogenate ◽  
Mouse Bioassay ◽  
Gpi Anchor ◽  
Glycosylphosphatidylinositol Anchor ◽  
Prion Infectivity ◽  
C Terminus ◽  
Prion Propagation

According to the protein-only hypothesis of prion propagation, prions are composed principally of PrPSc, an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrPC), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrPSc to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrPC. Removal of the GPI anchor from PrPSc requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrPSc in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrPSc, (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrPSc or on prion infectivity.

scholarly journals Multiple functions of inositolphosphorylceramides in the formation and intracellular transport of glycosylphosphatidylinositol-anchored proteins in yeast

2007 ◽  
Vol 74 ◽  
pp. 199-209 ◽  
Author(s):  
Régine Bosson ◽  
Andreas Conzelmann
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Fatty Acid ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Gpi Anchor ◽  
New Class ◽  
Golgi Transport ◽  
Lipid Moiety ◽  
Gpi Proteins

The mature sphingolipids of yeast consist of IPCs (inositolphosphorylceramides) and glycosylated derivatives thereof. Beyond being an abundant membrane constituent in the organelles of the secretory pathway, IPCs are also used to constitute the lipid moiety of the majority of GPI (glycosylphosphatidylinositol) proteins, while a minority of GPI proteins contain PI (phosphatidylinositol). Thus all GPI anchor lipids (as well as free IPCs) typically contain C26 fatty acids. However, the primary GPI lipid that isadded to newly synthesized proteins in the endoplasmic reticulum consists of a PI with conventional C16 and C18 fatty acids. A new class of enzymes is required to replace the fatty acid in sn-2 by a C26 fatty acid. Cells lacking this activity make normal amounts of GPI proteins but accumulate GPI anchors containing lyso-PI. As a consequence, the endoplasmic reticulum to Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. The GPI anchor containing C26 in sn-2 can further be remodelled by the exchange of diacylglycerol for ceramide. This process is also dependent on the presence of specific phosphorylethanolamine side-chains on the GPI anchor.

Mammalian GPI-anchor modifications and the enzymes involved

2020 ◽  
Vol 48 (3) ◽  
pp. 1129-1138 ◽  
Author(s):  
Yi-Shi Liu ◽  
Morihisa Fujita
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Mammalian Cells ◽  
Gpi Anchor ◽  
C Terminus ◽  
Lipid Moiety ◽  
Human Proteins

Glycosylphosphatidylinositol (GPI) is a glycolipid added to the C-terminus of a large variety of proteins in eukaryotes, thereby anchoring these proteins to the cell surface. More than 150 different human proteins are modified with GPI, and GPI-anchored proteins (GPI-APs) play critical roles in embryogenesis, neurogenesis, immunity, and fertilization. GPI-APs are biosynthesized in the endoplasmic reticulum (ER) and transported to the plasma membrane via the Golgi apparatus. During transport, GPI-APs undergo structural remodeling that is important for the efficient folding and sorting of GPI-APs. Asparagine-linked glycan-dependent folding and deacylation by PGAP1 work together to ensure that correctly folded GPI-APs are transported from the ER to the Golgi. Remodeling of the GPI lipid moiety is critical for the association of GPI-APs with lipid rafts. On the cell surface, certain GPI-APs are cleaved by GPI cleavage enzymes and released from the membrane, a key event in processes such as spermatogenesis and neurogenesis. In this review, we discuss the enzymes involved in GPI-AP biosynthesis and the fate of GPI-APs in mammalian cells, with a focus on the assembly, folding, degradation, and cleavage of GPI-APs.

Dimerization of the Antimicrobial Peptide Polyphemusin I into One Polypeptide Chain: Theoretical and Practical Consequences

2019 ◽  
Vol 35 (5) ◽  
pp. 36-41
Author(s):  
V.A. Zenin ◽  
E.G. Sadykhov ◽  
A.N. Fedorov
Keyword(s):  
Antimicrobial Peptides ◽  
Pore Formation ◽  
Lipid Membrane ◽  
Polypeptide Chain ◽  
Unique Identifier ◽  
Bacterial Cultures ◽  
In Series ◽  
The Russian Federation ◽  
Peptide Dimerization ◽  
Putative Mechanism

A strategy of sequential dimerization of monomers of antimicrobial peptides (AMPs) into one polypeptide chain has been implemented on the example of a beta-structural AMP polyphemusin I which is one of the most effective candidate for use as an antibiotic. The possible polyphemusin I monomer and dimer structures in lipid membrane were studied in this work via molecular modeling. To this end, these molecules were chemically synthesized so that the dimer represented two monomers connected in series into one polypeptide chain with a flexible linker. The antimicrobial effects of monomer and dimer were then tested on various bacterial cultures, and their similarity was shown. Therefore, we can conclude that the pore formation is not a putative mechanism of the polyphemusin I action. antimicrobial peptides, peptide dimerization, mechanism of antimicrobial action, polyphemusin The work was supported by the Ministry of Science and Higher Education of the Russian Federation (Project Unique Identifier RFMEFI57517X0151).

scholarly journals The C-terminal helix 9 motif in rat cannabinoid receptor type 1 regulates axonal trafficking and surface expression

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alexandra Fletcher-Jones ◽  
Keri L Hildick ◽  
Ashley J Evans ◽  
Yasuko Nakamura ◽  
Kevin A Wilkinson ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Secretory Pathway ◽  
Cannabinoid Receptor ◽  
Surface Expression ◽  
Surface Polarity ◽  
Time Resolved ◽  
Surface Stability ◽  
C Terminus ◽  
Time Resolved Imaging ◽  
Dual Mechanism

Cannabinoid type one receptor (CB1R) is only stably surface expressed in axons, where it downregulates neurotransmitter release. How this tightly regulated axonal surface polarity is established and maintained is unclear. To address this question, we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation in cultured rat hippocampal neurons. We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites. This dual mechanism is mediated by the CB1R C-terminus and involves the Helix 9 (H9) domain. Removal of the H9 domain increases secretory pathway delivery to dendrites and decreases surface stability. Furthermore, CB1RΔH9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT. Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process.

scholarly journals Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

2018 ◽  
Author(s):  
Karen Linnemannstöns ◽  
Pradhipa Karuna M ◽  
Leonie Witte ◽  
Jeanette Clarissa Kittel ◽  
Adi Danieli ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wnt Signaling ◽  
Signaling Pathways ◽  
Long Range ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Multivesicular Bodies ◽  
Phosphorylation Sites ◽  
Wnt Proteins ◽  
C Terminus

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion. However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6-RNAi mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. The latter accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies. Taken together, we show that a regulatory switch in Ykt6 fine-tunes sorting of Wnts in endosomes.

scholarly journals TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1256-1262 ◽  
Author(s):  
Thomas J. Girard ◽  
Elodee Tuley ◽  
George J. Broze
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Feedback Inhibition ◽  
Culture Media ◽  
Factor Viia ◽  
Factor Xa ◽  
Gpi Anchor ◽  
C Terminus ◽  
Before And After ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ.

scholarly journals Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A

1993 ◽  
Vol 295 (3) ◽  
pp. 813-819 ◽  
Author(s):  
A Varro ◽  
G J Dockray
Keyword(s):  
Ion Exchange ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Specific Activity ◽  
Brefeldin A ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Gastrin Cells ◽  
Tryptic Fragment ◽  
C Terminus

The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory granule; cleavage is known to occur in the secretory granule which suggests that it occurs after phosphorylation.

scholarly journals Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall.

1995 ◽  
Vol 128 (3) ◽  
pp. 333-340 ◽  
Author(s):  
C F Lu ◽  
R C Montijn ◽  
J L Brown ◽  
F Klis ◽  
J Kurjan ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Secretory Pathway ◽  
Molecular Size ◽  
Cross Linking ◽  
Cell Contact ◽  
Gpi Anchor ◽  
Adhesion Protein ◽  
Glycosyl Phosphatidylinositol ◽  
Cross Linkage

The cell adhesion protein alpha-agglutinin is bound to the outer surface of the Saccharomyces cerevisiae cell wall and mediates cell-cell contact in mating. alpha-Agglutinin is modified by addition of a glycosyl phosphatidylinositol (GPI) anchor as it traverses the secretory pathway. The presence of a GPI anchor is essential for cross-linking into the wall, but the fatty acid and inositol components of the anchor are lost before cell wall association (Lu, C.-F., J. Kurjan, and P. N. Lipke, 1994. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol. Cell. Biol. 14:4825-4833). Cell wall association of alpha-agglutinin was accompanied by an increase in size and a gain in reactivity to antibodies directed against beta 1,6-glucan. Several kre mutants, which have defects in synthesis of cell wall beta 1,6-glucan, had reduced molecular size of cell wall alpha-agglutinin. These findings demonstrate that the cell wall form of alpha-agglutinin is covalently associated with beta 1,6-glucan. The alpha-agglutinin biosynthetic precursors did not react with antibody to beta 1,6-glucan, and the sizes of these forms were unaffected in kre mutants. A COOH-terminal truncated form of alpha-agglutinin, which is not GPI anchored and is secreted into the medium, did not react with the anti-beta 1,6-glucan. We propose that extracellular cross-linkage to beta 1,6-glucan mediates covalent association of alpha-agglutinin with the cell wall in a manner that is dependent on prior addition of a GPI anchor to alpha-agglutinin.

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