title: 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.
authors.affiliation: Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4480, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Arnaoutova
authors.forename: Angela M
authors.initials: AM
authors.lastname: Smith
authors.forename: Leigh C
authors.initials: LC
authors.lastname: Coates
authors.forename: Juanita C
authors.initials: JC
authors.lastname: Sharpe
authors.forename: Savita
authors.initials: S
authors.lastname: Dhanvantari
authors.forename: Chris R
authors.initials: CR
authors.lastname: Snell
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Birch
authors.forename: Y Peng
authors.initials: YP
authors.lastname: Loh
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D017434:Protein Structure, Tertiary; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D000324:Adrenocorticotropic Hormone; D043484:Proprotein Convertases; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases