Functional asymmetry within the Sec61p translocon.

Area of Science:

• Molecular Biology
• Cell Biology
• Protein Translocation

Background:

• The Sec61 translocon facilitates protein transport across membranes.
• It also integrates hydrophobic segments into the membrane via a lateral gate.
• A central pore constriction influences membrane integration hydrophobicity.

Purpose of the Study:

• Investigate the role of the translocon's central constriction in hydrophobic segment integration.
• Determine the impact of residue hydrophobicity at the constriction on integration.
• Explore functional asymmetry within the Sec61 translocon during membrane insertion.

Main Methods:

• Site-directed mutagenesis of yeast Sec61p.
• Analysis of hydrophobic (H) segment integration efficiency.
• Scanning mutagenesis to map positional effects within H segments.

Main Results:

• Mutations at the constriction altered hydrophobicity requirements for integration.
• H-segment integration showed asymmetry, with preferential insertion in the N-terminal half.
• Integration was reduced when H-segment hydrophobic residues aligned with the pore constriction.
• Mutating constriction residues to glycine or serine abolished position effects and asymmetry.
• Asymmetry persisted when constriction residues were mutated to alanine.

Conclusions:

• Translocon interior hydrophobicity, particularly at the central constriction, critically influences membrane protein insertion.
• Hydrophobic segment integration is not solely dependent on intrinsic hydrophobicity but also on translocon environment.
• The contribution of hydrophobic residues is context-dependent and exhibits cooperativity.