Proprotein convertase PC3 is not a transmembrane protein

Area of Science:

• Molecular Cell Biology
• Endocrinology
• Protein Biochemistry

Background:

• Proprotein convertase PC3 (PC1) is crucial for processing peptide hormones in endocrine cells.
• PC3 was previously thought to be a peripheral membrane protein due to its lack of hydrophobic segments.
• Recent studies suggested PC3 might be a transmembrane protein with charged amino acids in its membrane-spanning region.

Purpose of the Study:

• To definitively determine the membrane topology of proprotein convertase PC3.
• To investigate whether PC3 functions as a transmembrane protein within the regulated secretory pathway.
• To clarify the implications of PC3's topology for its granule sorting mechanism.

Main Methods:

• Analysis of PC3 membrane topology using alkaline extraction to assess membrane integration.
• Utilized C-terminal tags for phosphorylation (cytosol exposure) and glycosylation (ER lumen exposure).
• Assayed protease sensitivity in permeabilized cells to confirm protein localization.

Main Results:

• The C-terminus of PC3 was translocated across the endoplasmic reticulum membrane.
• PC3's proposed transmembrane segment did not halt polypeptide translocation in a stop-transfer assay.
• Evidence indicates PC3 is not a transmembrane protein, contrary to recent hypotheses.

Conclusions:

• Proprotein convertase PC3 is not a transmembrane protein.
• The findings necessitate a re-evaluation of PC3's mechanism for sorting into secretory granules.
• This study impacts the understanding of the membrane topology of related proteins like PC2 and carboxypeptidase E.