Question 1

What signal sequences does a GPI-anchored protein contain?

Accepted Answer

A GPI-anchored protein contains two signal sequences: an N-terminal ER signal and a C-terminal GPI-anchoring signal. The ER signal directs the protein to the endoplasmic reticulum for translocation. The GPI-anchoring signal, located after the last amino acid of the target protein, is 20-30 amino acids long with hydrophilic and hydrophobic regions that direct GPI attachment.

Question 2

How does GPI transamidase modify the target protein?

Accepted Answer

GPI transamidase is a five-subunit ER-resident enzyme complex that cleaves the target protein between the ω and ω+1 residues of the GPI-attachment signal sequence. This cleavage generates an intermediate comprising the enzyme complex and target protein. The complex then transfers the C-terminal of the protein onto a pre-assembled GPI anchor embedded in the ER membrane's luminal leaflet.

Question 3

What is the structural composition of a GPI anchor?

Accepted Answer

A GPI anchor consists of hydrophobic and hydrophilic portions. The hydrophobic portion comprises phosphatidylinositol with acyl chains that anchor the protein to the membrane. The hydrophilic part contains polar groups including phosphoethanolamine, glucosamine, mannose residues, and phosphate groups. Phosphoethanolamine interacts with the target protein's C-terminal.

Question 4

How does GPI-anchoring differ from transmembrane protein insertion?

Accepted Answer

Unlike transmembrane proteins that span the lipid bilayer, GPI-anchored proteins are only covalently bound to the ER membrane through a glycolipid anchor. This allows GPI-anchored proteins to move freely through the hydrophobic interior of the lipid bilayer. After assembly on the ER membrane, they are transported to the plasma membrane's exoplasmic leaflet.

Question 5

What happens to the ER signal peptide during GPI-anchored protein synthesis?

Accepted Answer

As the protein descends through the Sec61 channel during translocation, the signal peptidase complex cleaves off the N-terminal ER signal. This cleavage occurs early in translocation, converting the preproprotein into a proprotein. The proprotein continues translocation until the C-terminal GPI-anchoring signal approaches the channel exit, where GPI transamidase acts.

Question 6

Where are GPI-anchored proteins localized in the cell?

Accepted Answer

GPI-anchored proteins are primarily present on the exoplasmic leaflet of the plasma membrane, where they face the cell's exterior. These proteins associate with membrane microdomains or membrane rafts, which are sites of dynamic cellular functions including signal transduction, receptor activation, and regulation of membrane traffic. They also contribute to establishing apical polarity in epithelial cells.

Question 7

What are the two translocation pathways for GPI-anchored protein precursors?

Accepted Answer

GPI-anchored protein precursors can use two translocation routes into the ER lumen. The prion protein follows the post-translational route, where translocation occurs after synthesis. The CD59 protein uses the signal recognition particle or SRP-dependent cotranslational pathway, where translocation occurs during protein synthesis. Both pathways require the N-terminal ER signal sequence.

GPI Anchoring of Proteins in the ER Membrane

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