Glucocorticoid-regulated trafficking of mouse mammary tumor virus proteins in permeabilized hepatoma cells. Requirements of intracellular membrane transport for maturation of the cytoplasmic phosphorylated polyprotein.
Glucocorticoids coincidentally regulate the localization of mouse mammary tumor virus (MMTV) glycoproteins and maturation of viral phosphoproteins in viral infected rat hepatoma cells. To test for a functional interaction between MMTV transmembrane glycoproteins and cytoplasmic phosphoproteins, the bacterial cytolysin streptolysin-O was utilized to selectively permeabilize the plasma membrane and reconstitute exocytic trafficking. Streptolysin-O-permeabilized M1.54 cells pretreated with glucocorticoids retained the capability for proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins as determined by immunoprecipitation and immunofluorescence microscopy. The efficient maturation of MMTV phosphoproteins indicated that these viral proteins are properly transported near or to the plasma membrane in permeabilized cells. These maturation events in semi-intact cells were dependent on the addition of cell cytosol and were specifically inhibited by the membrane impermeant GTP analog guanosine 5'3-O-(thio)triphosphate, an agent known to impede vesicular transport of membrane proteins, but which has not previously been shown to alter cytoplasmic protein maturation or transport. The addition of anti-MMTV antibodies directed against the cytoplasmic domain of the glycoprotein precursor to transport competent semi-intact M1.54 cells resulted in the dramatic inhibition of both MMTV glycoprotein and phosphoprotein maturation. These results were not obtained using either preimmune sera or antiserum specific for the luminal portion of the glycoprotein precursor. Our findings suggest that the functional interaction of cytosolic MMTV phosphoproteins with the cytoplasmic domain of the viral membrane glycoprotein is required for the efficient transport and processing of each class of proteins in glucocorticoid-treated cells and provides the first evidence for the involvement of vesicular transport in the delivery and maturation of cytoplasmic viral proteins at the plasma membrane or the pericellular region.
Department of Molecular and Cell Biology, University of California at Berkeley 94720.
S R
SR
Kain
T I
TI
Jen
G L
GL
Firestone
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D020126:Brefeldin A; D002463:Cell Membrane Permeability; D002999:Clone Cells; D003517:Cyclopentanes / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008114:Liver Neoplasms, Experimental; D008324:Mammary Tumor Virus, Mouse / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D010750:Phosphoproteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D013301:Streptolysins; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*
D000998:Antiviral Agents; D001426:Bacterial Proteins; D003517:Cyclopentanes; D010750:Phosphoproteins; D013301:Streptolysins; D014764:Viral Proteins; C025964:streptolysin O; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D003907:Dexamethasone; D016650:Fluorescein-5-isothiocyanate