The Prohormone Processing Enzyme PC3 Is a Lipid Raft-Associated Transmembrane Protein

Abstract

The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.

Abnormal sterols in cholesterol-deficiency diseases cause secretory granule malformation and decreased membrane curvature

“…Anti-CgA polyclonal IgG was previously described (Arnaoutova et al, 2003). Anti-p115 mouse IgG is from BD Transduction Laboratories (San Jose, CA).…”

The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

“…The presence of full-length PC1/3 with its C-terminal region intact is required for the processing of prorenin to renin in granules in AtT-20 cells (Jutras et al 1997). Without its lipid-binding C-terminal region (Jutras et al 2000, Arnaoutova et al 2003, PC1/3 is not sorted to the regulated secretory pathway, and renin is secreted constitutively. PC2 also contains an alpha-helical, lipid-binding C-terminal tail that directs it to the regulated pathway (Assadi et al 2004), thus enabling it to act as a membrane-bound receptor/ tether.…”

“…Sequence similarity suggests that GLP-2 is also an alpha-helix, and it has been suggested that the structure of the entire proglucagon molecule may form a 'trimer' of alpha helices (Dey et al 2004). Conserved alpha-helix structures within the amino-terminal end of prosomatostatin (Mouchantaf et al 2001) and the C-terminal ends of PC1/3 (Jutras et al 2000, Arnaoutova et al 2003, PC2 (Assadi et al 2004), PC5/6a ) and CPE (Dhanvantari et al 2003a are critical in targeting these proteins to secretory granules. Additionally, the sequence of glucagon contains an internal pair of basic amino acids, Arg17Arg18, which does not serve as a PC1/3 or PC2 cleavage site (Bataille 2007).…”

Modulation of Secretory Granule-targeting Efficiency by Cis and Trans Compounding of Sorting Signals

“…These include paired basic residues that may interact with granule-resident PC enzymes (14, 24 -26), N-terminal disulfide-bound loops (27,28), and other domains that mediate sorting by binding to caboxypeptidase E (2), calcium-binding domains that lead to aggregation (reviewed in Ref. 8), and ␣-helices that either traverse or interact with membranes (3,(5)(6)(7). Finally, proteins may contain more than one single type of sorting domain so that analyses in which any one of these is eradicated will not eliminate sorting in the context of the native protein.…”

“…This process, called regulated secretion, depends on a highly selective triage of proteins within or just after the TGN. Retention in dense core secretory granules is not a random process and granulesorted proteins must possess unique properties or must interact with other cellular components in a unique way to end up in this organelle (1)(2)(3)(4)(5)(6)(7).…”