The C-terminus of prohormone convertase 2 is sufficient and necessary for Raft association and sorting to the regulated secretory pathway.
Prohormone convertase 2 (PC2) is a member of the subtilisin family of proteases involved in prohormone maturation in the granules of the regulated secretory pathway (RSP). It has been suggested that targeting of this enzyme to the RSP is dependent on its association with lipid rafts in membranes at the trans-Golgi network. Here, we investigate the orientation of PC2 in granule membranes and the role of the C-terminus in sorting of the enzyme to the RSP. Molecular modeling and circular dichroism showed that this domain of PC2 forms an alpha-helix and inserts into artificial membranes. Furthermore, we show that the C-terminus of PC2 can be biotinylated at the C-terminus in intact chromaffin granules, indicating that it is a transmembrane protein. To determine if the PC2 C-terminus is necessary for raft association and sorting, we transfected a chimera of CPEDelta15 (carboxypeptidase E without the last 15 residues) and the last 25 residues of PC2 (CPEDelta15-PC2), and a truncated PC2 mutant with the last 6 residues deleted (PC2Delta6) into Neuro2a cells. Whereas CPEDelta15 was not raft-associated or sorted to the RSP, addition of the 25 residues of PC2 C-terminus to CPEDelta15 restored raft association and localization to the RSP granules, as determined by immunocytochemistry. Deletion of the last 6 residues of PC2 eliminated lipid raft association and sorting of PC2Delta6 to the RSP. These results showed that the PC2 C-terminus confers raft association and is sufficient and necessary for sorting PC2 to the RSP.
Assadi
Masoumeh
M
Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4480, USA.
Sharpe
Juanita C
JC
Snell
Christopher
C
Loh
Y Peng
YP
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D045706:Proprotein Convertase 2 / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D045706:Proprotein Convertase 2