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

M Blazquez, C Thiele, W B Huttner, K Docherty, K I J Shennan

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57 Citations (Scopus)

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 (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 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.

Original languageEnglish
Pages (from-to)843-852
Number of pages10
JournalBiochemical Journal
Volume349
Publication statusPublished - 2000

Keywords

  • lipid rafts
  • protein targeting
  • regulated secretion
  • sorting mechanisms
  • TRANS-GOLGI NETWORK
  • DISULFIDE-BONDED LOOP
  • CARBOXYPEPTIDASE-E
  • CHROMOGRANIN-B
  • CHROMAFFIN GRANULES
  • KEX2-RELATED PROTEASES
  • PC2
  • IDENTIFICATION
  • PROTEINS
  • CELLS

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