Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway

Author:

BLÁZQUEZ Mercedes1,THIELE Christoph2,HUTTNER Wieland B.2,DOCHERTY Kevin1,SHENNAN Kathleen I. J.1

Affiliation:

1. Department of Molecular and Cell Biology, Institute of Medical Sciences. University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, U.K.

2. Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, Heidelberg 69120, Germany

Abstract

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 (proPC245–84) 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 proPC245–84. Rather, proPC245–84 was shown to bind to protein-free liposomes in a pH- and Ca2+-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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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