Interactions between Sec complex and prepro-alpha-factor during posttranslational protein transport into the...

Area of Science:

• Cell Biology
• Molecular Biology
• Protein Translocation

Background:

• Protein translocation across the endoplasmic reticulum (ER) membrane is crucial for protein folding and function.
• The Sec complex, comprising Sec61p and Sec62p/63p, mediates this process in yeast.
• Understanding the precise interactions within the Sec complex is key to elucidating translocation mechanisms.

Purpose of the Study:

• To investigate the initial signal sequence recognition step by the Sec complex.
• To identify the transmembrane segments of Sec61p that form the translocation pore.
• To characterize the interaction between the translocating polypeptide and the Sec61p pore.

Main Methods:

• Photo-cross-linking experiments were employed to map protein-protein interactions.
• Photoreactive probes were incorporated into prepro-alpha-factor (ppalphaF) at specific positions.
• Analysis of cross-linked products identified contact sites between ppalphaF and the Sec complex components.

Main Results:

• The signal sequence of ppalphaF simultaneously contacts both Sec61p and Sec62p, indicating a single recognition site.
• No evidence was found for signal sequence contact with two Sec61p molecules.
• Multiple transmembrane segments of Sec61p were identified as lining the translocation pore.
• Neighboring positions of the mature ppalphaF showed similar interactions with Sec61p, suggesting low affinity for the pore lining.

Conclusions:

• The yeast Sec complex utilizes a unified recognition site for signal sequences involving Sec61p and Sec62p.
• The translocation pore is formed by multiple Sec61p transmembrane segments.
• These pore-lining segments exhibit minimal affinity for the translocating polypeptide, facilitating passage.