Protein O-mannosyltransferases associate with the translocon to modify translocating polypeptide chains

Area of Science:

• Cellular biology
• Protein biochemistry
• Glycoscience

Background:

• O-mannosylation and N-glycosylation are crucial protein modifications initiated in the endoplasmic reticulum (ER).
• Protein translocation and N-glycosylation are coordinated at the translocon-oligosaccharyltransferase (OST) complex.
• Previously, O-mannosylation was thought to occur post-translocation, but recent findings suggest it can happen during prolonged ER residence for misfolded proteins.

Purpose of the Study:

• To investigate the role of protein O-mannosyltransferases (PMTs) in protein translocation.
• To determine if PMTs interact with the translocation machinery.
• To establish an in vitro system for studying co-translational O-mannosylation.

Main Methods:

• Co-immunoprecipitation to demonstrate in vivo association of Pmt1-Pmt2 with OST, Sec61, and Sec63 complexes.
• Mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants.
• Development of a microsomal in vitro system for translation, translocation, and O-mannosylation.

Main Results:

• Demonstrated in vivo association of Pmt1-Pmt2 with the OST complex, Sec61, and Sec63.
• Mass spectrometry revealed coordinated interplay between PMTs and OST.
• Established a functional in vitro system showing PMTs mannosylate proteins during translocation into microsomes.

Conclusions:

• Protein O-mannosylation, mediated by PMTs, is coordinated with protein translocation at the ER membrane.
• The developed in vitro system allows for mechanistic studies of co-translational O-mannosylation.
• This work challenges previous assumptions and provides new insights into the timing and location of O-mannosylation.