Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway.
Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggregation and membrane association during its transit through the regulated secretory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate this further, we determined the binding properties of a peptide containing amino acids 45-84 of proPC2 (proPC2(45-84)) to trans-Golgi network/granule-enriched membranes from the AtT20 cell line. Removal of peripheral membrane proteins or hydrolysis of integral membrane proteins did not affect the binding properties of proPC2(45-84). Rather, proPC2(45-84) was shown to bind to protein-free liposomes in a pH- and Ca(2+)-dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we used chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depleting detergent, failed to extract PC2 from the membranes, whereas chromogranin A (CgA) was removed. Treatment of the membranes with Triton X-100 yielded a low-density detergent-insoluble fraction enriched in PC2, but not CgA. The detergent-insoluble fraction also contained glycoprotein III, known to be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid rafts in the membrane and its sorting into the regulated secretory pathway.
Blázquez
M
M
Department of Molecular and Cell Biology, Institute of Medical Sciences. University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, U.K.
Thiele
C
C
Huttner
W B
WB
Docherty
K
K
Shennan
K I
KI
D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents; D006056:Golgi Apparatus / Q000378:metabolism; D008051:Lipid Bilayers; D008081:Liposomes; D045706:Proprotein Convertase 2; D013381:Subtilisins / Q000378:metabolism*
D003902:Detergents; D008051:Lipid Bilayers; D008081:Liposomes; D002784:Cholesterol; D013381:Subtilisins; D045706:Proprotein Convertase 2