O-mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation

Area of Science:

• Cellular Biology
• Protein Folding and Degradation
• Endoplasmic Reticulum Quality Control

Background:

• Secretory proteins misfolding in the endoplasmic reticulum (ER) are targeted for cytosolic degradation.
• Mechanisms distinguishing folding intermediates from irreversibly misfolded proteins remain unclear.
• Covalent modification of misfolded proteins within the ER prior to export is investigated.

Purpose of the Study:

• To investigate whether misfolded secretory proteins undergo covalent modification in the ER before export.
• To elucidate the role of O-mannosylation in the ER-associated degradation pathway.
• To determine the impact of modifications on the fate of misfolded proteins.

Main Methods:

• Utilized yeast microsomes and intact yeast cells for studying protein modification.
• Investigated O-mannosylation of mutant alpha-factor precursor using protein O-mannosyl transferase 2 (Pmt2p).
• Assessed the effect of ATP, cytosol, and Sec61p on O-mannosylation and protein export.

Main Results:

• Misfolded alpha-factor precursor, but not wild-type, was progressively O-mannosylated by Pmt2p in vitro and in vivo.
• O-mannosylation was enhanced under ER export conditions (ATP, cytosol) and required Sec61p.
• O-mannosylated misfolded protein showed stability, protease resistance, and association with Sec61p, hindering ER export.
• Deletion of PMT2 enhanced turnover of misfolded alpha-factor precursor.

Conclusions:

• Prolonged ER residence of misfolded proteins leads to O-mannosylation of exposed sites.
• This modification impedes ER export at a post-targeting stage, interfering with Sec61-mediated disposal.
• A critical time window exists for misfolded protein removal before irreversible modification.