potassium-acetate and sodium-carbonate

Production of interleukin-12 (IL-12), a heterodimer of p35 and p40 subunits, is limited by p35 expression. A long and a short murine p35 mRNA potentially encoding proteins differing in pre-sequence size are produced. Increased pre-sequence size could convert a cleaved signal peptide to an uncleaved signal peptide, raising the possibility that a membrane-bound form of p35 is produced. The intracellular localization of the p35 encoded by each mRNA isoform was determined by constructing cDNAs containing the long or short p35 cDNA isoform fused in-frame to a cDNA encoding green fluorescent protein (GFP). After transfection of a CV-1 African green monkey kidney cell line with the constructs, confocal microscopy and immunoblotting of extracted microsomal membranes demonstrated that the p35-GFP fusion protein encoded by the long or short mRNA accumulates in the Golgi apparatus as an endoglycosidase H-sensitive glycosylated integral membrane protein. In contrast, a p40-GFP fusion protein accumulates in the Golgi apparatus as a soluble protein. Since assembly of the p35 and p40 subunits to form bioactive IL-12 occurs in the ER, release of membrane-tethered IL-12 by proteolytic cleavage in a late Golgi or post-Golgi compartment may represent an as yet unidentified level at which bioactive IL-12 secretion is regulated.

Topics: Animals; Carbonates; Cell Membrane; Chlorocebus aethiops; Cloning, Molecular; Dimerization; DNA, Complementary; Glycoside Hydrolases; Glycosylation; Golgi Apparatus; Green Fluorescent Proteins; Interleukin-12; Interleukin-12 Subunit p35; Luminescent Proteins; Mice; Microscopy, Confocal; Microsomes, Liver; Peptides; Potassium Acetate; Protein Isoforms; Protein Sorting Signals; Protein Subunits; Recombinant Fusion Proteins; RNA, Messenger; Transfection