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Intracellular localization of the p35 subunit of murine IL-12
Hema Vaidyanathan1, You Zhou, Thomas M Petro
1Center for Biological Chemistry, University of Nebraska, Lincoln, NE 68588, USA.
Abstract:
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.
Insights
Interleukin-12 (IL-12) production is limited by p35 expression. This study found that p35 protein accumulates in the Golgi as a membrane protein, suggesting a new regulation point for IL-12 secretion.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Interleukin-12 (IL-12) is a critical cytokine for immune responses, composed of p35 and p40 subunits.
- IL-12 production is often limited by the expression of the p35 subunit.
- Two murine p35 mRNA isoforms exist, potentially differing in signal peptide size and affecting protein localization.
Purpose of the Study:
- To investigate the intracellular localization of p35 protein isoforms.
- To determine if p35 can be membrane-bound and how this impacts IL-12 assembly and secretion.
- To identify potential novel regulatory mechanisms for bioactive IL-12 secretion.
Main Methods:
- Constructing cDNA fusions of long/short p35 isoforms with green fluorescent protein (GFP).
- Transfecting CV-1 cells with p35-GFP constructs.
- Utilizing confocal microscopy and immunoblotting of microsomal membranes to analyze protein localization and modification.
Main Results:
- Both long and short p35 isoforms fused to GFP accumulate in the Golgi apparatus.
- The p35-GFP fusion proteins are glycosylated and integral membrane proteins, sensitive to endoglycosidase H.
- In contrast, a p40-GFP fusion protein localizes to the Golgi as a soluble protein.
Conclusions:
- The p35 subunit integrates into the membrane, unlike the soluble p40 subunit.
- IL-12 subunit assembly occurs in the ER, but p35's membrane integration suggests post-ER processing.
- Proteolytic cleavage in late Golgi or post-Golgi compartments may regulate the release and secretion of membrane-tethered IL-12, representing a novel regulatory step.
