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Eunice Kennedy Shriver National Institute of Child Health and Human Development - Section on Cellular Neurobiology - Dr. Peng Loh Lab
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Sorting of Processing Enzymes to the Regulated Secretory Pathway

œ-Helices and Lipid Rafts

After being targeted to the immature secretory granule from the TGN, prohormones are processed through the actions of the prohormone convertase enzymes, PC1 (also known as PC3) or PC2. These convertase enzymes are expressed exclusively in neural and endocrine cells, and cleave prohormones at pairs of basic amino acids. Following this endoproteolytic cleavage, the C-terminal basic residues are trimmed by the enzymatically active form of CPE to yield mature, bioactive peptide hormones.

Since the processing enzymes (PC1, PC2 and CPE) are active only within the high Ca 2+, low pH environment of the secretory granule, they must be sorted along with their prohormone substrates to the regulated secretory pathway. These enzymes may share a common mechanism for sorting that involves lipid microdomains, or "rafts". Rafts are microdomains within cell membranes that are enriched in saturated lipids, such as sphingolipids, and cholesterol. Rafts may serve to cluster protein complexes, such as those involved in signal transduction, thereby facilitating signaling. Using a number of techniques such as pulse-chase, subcellular fractionation, lipid analysis by quantitative TLC and fluorescence immunocytochemistry, we have demonstrated that cholesterol is the most abundant lipid in the membranes of the TGN and secretory granules, and that the function of CPE as a sorting receptor is dependent on cholesterol. Raft association of CPE likely facilitates its interaction with its prohormone cargo. We also found that sorting of PC1 is mostly dependent on a transmembrane domain (aa617-638). This sequence is raft associated and sufficient to target PC1 (its native molecule) or a reporter protein to the regulated secretory pathways.

Molecular modeling of these enzymes has revealed an amphipathic alpha helix within the C-terminal domains of these proteins. These regions have recently been shown to be raft associated and to mediate the targeting of PC1, PC2 and CPE to the RSP. The amphipathic alpha helical region of the processing enzymes may therefore serve dual functions: as a membrane anchor within lipid microdomains of the TGN and secretory granules, and as a sorting domain.