Name: how gpi-linked proteins reach the membrane
Uploaded: 2023-04-30T10:12:30.000Z
Duration: 1 min 29 s
Channel: JoVE

A protein destined for glycosylphosphatidylinositol or GPI-anchoring contains an N-terminal ER signal and a C-terminal GPI-anchoring signal.
Since the GPI-anchoring occurs in the ER lumen, the target protein must be translocated across the ER membrane.
As the protein descends down the Sec61 channel, the signal peptidase complex cleaves off its ER signal.
The translocation continues until the C-terminal GPI-anchoring signal is about to exit the channel.
At this point, GPI transamidase &#8211; an ER-resident enzyme complex, cleaves the target protein.
The cleavage generates an intermediate comprising the enzyme complex and the target protein, leaving the GPI-anchoring signal sequence in the membrane.
This intermediate transfers the target protein onto a pre-assembled GPI anchor, which is a complex glycolipid embedded in the luminal leaflet of the ER membrane.
Unlike the transmembrane proteins, the resultant GPI-anchored protein is only covalently bound to the ER membrane and can move freely through the hydrophobic interior of the lipid bilayer.
After their assembly on the ER membrane, GPI-anchored proteins are transported to the plasma membrane to face the cell&#39;s exterior.

gpi anchoring of proteins in the er membrane

GPI Anchoring of Proteins in the ER Membrane

GPI-anchoring is a post-translational, reversible protein modification that is ubiquitous in eukaryotes. Such proteins are primarily present on the exoplasmic leaflet of the plasma membrane.
GPI-anchor structure 
A sequence of 11 enzymatic reactions results in the synthesis of the complete GPI anchor consisting of a hydrophobic and a hydrophilic portion. The hydrophobic portion comprises phosphatidylinositol, while the hydrophilic part comprises polar groups like phosphoethanolamine, glucosamine, mannose residues, and phosphate groups. The phosphoethanolamine interacts with the C-terminal of the target protein, while the acyl chains of phosphatidylinositol act as the membrane anchors.
Translocation of the precursor protein
The synthesis of a lipid-anchored protein has precursor stages. The preproprotein comprises two signal sequences, one at each end of the target protein. The ER signal sequence is present at the N-terminal, while the GPI-attachment signal is present towards the C terminal following the last amino acid of the target protein. The start position of the GPI-attachment signal is termed &#9077;+1 in the preproprotein. It is a 20-30 amino acid long sequence with 10 hydrophilic residues followed by 20 hydrophobic residues. The mechanism of translocation of the preproprotein into the ER lumen depends on the type of target protein. For example, while the prion protein takes the post-translational route, the CD59 protein uses the signal recognition particle or SRP-dependent cotranslational pathway.
Transfer of the protein to the GPI anchor
After the N-terminal signal is cleaved from the preproprotein, it results in the formation of a proprotein. This proprotein is acted upon by GPI transamidase, a five subunit complex that cleaves between &#9077; and the &#9077;+1 residue of the GPI-attachment signal sequence. Additionally, this complex transfers the C-terminal of the protein onto the pre-assembled GPI-anchor.
Role of GPI-anchored proteins in the cells
The GPI-anchored proteins are associated with membrane microdomains or membrane rafts that are sites of dynamic cellular functions like signal transduction, receptor activation, and regulation of membrane traffic. Also, they contribute to the establishment of apical polarity like in the epithelial cell type by associating with the apical surface.

GPI-anchoring is a post-translational, reversible protein modification that is ubiquitous in eukaryotes. Such proteins are primarily present on the ...

gpi-anchoring-of-proteins-in-the-er-membrane

Education

JoVE Core

Cell Biology

Unit 1

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

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KEY TERMS AND CONCEPTS

how gpi-linked proteins reach the membrane

GPI-linked proteins are made through a post-translational modification that helps place them on the outer side of the plasma membrane in eukaryotic cells. The GPI, or glycosylphosphatidylinositol, anchor is reversible and is built through 11 enzyme-driven steps. It includes a hydrophobic part and a hydrophilic part.

The hydrophobic region contains phosphatidylinositol. The hydrophilic region includes phosphoethanolamine, glucosamine, mannose residues, and phosphate groups. Phosphoethanolamine connects with the protein’s C-terminal end. The acyl chains of phosphatidylinositol hold the anchor in the membrane.

A GPI-anchored protein starts as a precursor called a preproprotein. This precursor has two signal sequences, one at each end of the target protein. The ER signal sequence is at the N-terminal end. The GPI-attachment signal is near the C-terminal end, after the last amino acid of the target protein.

The start of the GPI-attachment signal is called ω+1 in the preproprotein. This signal is about 20 to 30 amino acids long. It contains about 10 hydrophilic residues followed by about 20 hydrophobic residues. How the preproprotein enters the ER lumen depends on the protein. The prion protein uses a post-translational route. CD59 uses an SRP, or signal recognition particle, dependent cotranslational pathway.

After the N-terminal signal is removed, the protein becomes a proprotein. GPI transamidase, a five-subunit complex, then acts on it. This complex cuts between the ω and ω+1 residues of the GPI-attachment signal. It also transfers the protein’s C-terminal end onto the preassembled GPI anchor.

GPI-anchored proteins often sit in membrane microdomains, also called membrane rafts. These regions support cell activities such as signal transduction, receptor activation, and membrane traffic control. They also help establish apical polarity in epithelial cells by associating with the apical surface.

How GPI-Linked Proteins Reach the Membrane

how-gpi-linked-proteins-reach-the-membrane

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key terms and concepts

Learn how GPI-linked proteins are built, targeted to the ER, and attached to the membrane with GPI transamidase and membrane raft roles.

How GPI-Linked Proteins Reach the Membrane | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

How GPI-Linked Proteins Reach the Membrane

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