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Facultative polypeptide translocation allows a single mRNA to encode the secreted and cytosolic forms of plasminogen
D Belin1, A Wohlwend, W D Schleuning
1Département de Pathologie, University of Geneva Medical Center, Switzerland.
Abstract:
Two forms of plasminogen activators inhibitor 2 (PAI-2) are synthesized by human and murine monocytes/macrophages: one accumulates in the cytosol, while the other is translocated into the endoplasmic reticulum, glycosylated and secreted. We show here that a single mRNA encodes both forms of PAI-2. Firstly, a single PIA-2 mRNA was detected by Northern blot hybridization and by RNase protection. Secondly, transfection of a PAI-2 cDNA led to the synthesis of both forms of PAI-2. Finally, in vitro translation of an mRNA transcript of the PAI-2 cDNA in the presence of microsomal membranes generated two topologically distinct forms of PAI-2. The cytosolic and secreted forms of PAI-2 do not result from the use of two translation start sites, since their synthesis initiates at the same AUG, in a sequence context that is conserved between the human and murine genes. Thus, the accumulation of one polypeptide into two topologically distinct cellular compartments can be achieved by facultative translocation.
Insights
A single messenger RNA (mRNA) encodes both cytosolic and secreted forms of plasminogen activator inhibitor 2 (PAI-2). This facultative translocation mechanism allows one PAI-2 polypeptide to localize to distinct cellular compartments.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Monocytes/macrophages synthesize two forms of plasminogen activator inhibitor 2 (PAI-2).
- One PAI-2 form resides in the cytosol, while the other is secreted after ER translocation and glycosylation.
Purpose of the Study:
- To investigate the molecular basis for the synthesis of two distinct PAI-2 forms from a single gene.
- To determine if a single mRNA transcript encodes both cytosolic and secreted PAI-2.
Main Methods:
- Northern blot hybridization and RNase protection assays to detect PAI-2 mRNA.
- Transfection of PAI-2 cDNA to assess protein synthesis.
- In vitro translation assays with microsomal membranes.
Main Results:
- A single PAI-2 mRNA transcript was detected, encoding both protein forms.
- Transfection and in vitro translation confirmed the synthesis of both cytosolic and secreted PAI-2.
- Both forms initiate translation from the same AUG start site, indicating facultative translocation.
Conclusions:
- A single mRNA and translation start site are responsible for generating two distinct PAI-2 protein forms.
- Facultative translocation dictates the cellular localization and fate of PAI-2.
- This mechanism provides cellular flexibility in PAI-2 distribution and function.