Disruptions in intracellular membrane trafficking and structure preclude the glucocorticoid-dependent maturation of mouse mammary tumor virus proteins in rat hepatoma cells.
We have previously shown that glucocorticoids regulate the trafficking and processing of mouse mammary tumor virus (MMTV) proteins in viral-infected M1.54 rat hepatoma cells. To examine the role of intracellular membrane integrity on MMTV protein maturation, brefeldin A (BFA) was utilized to disrupt membrane flow between the endoplasmic reticulum and Golgi. Immunoprecipitation and immunofluorescence microscopy revealed that in the presence of dexamethasone, BFA inhibited the proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins. Glycosidase digestion and inhibitors of protein glycosylation confirmed that the observed differences in apparent sizes of MMTV glycoprotein products are due to BFA-induced changes in oligosaccharide processing. BFA treatment inhibited the proteolytic processing of the MMTV phosphoprotein precursor, which normally associates with the cytoplasmic face of intracellular membranes. Similarities in salt extraction efficiency revealed that BFA did not affect the membrane affinity of the uncleaved phosphorylated precursor. In a complementary approach, proteolytic processing of the phosphorylated polyprotein did not occur in glucocorticoid-treated HTC cells transfected with a mutant MMTV provirus encoding a normal phosphorylated precursor, but which express a truncated MMTV glycoprotein missing its transmembrane domain and cytoplasmic tail. These results suggest that the MMTV glycoproteins and phosphoproteins may interact at a late step in the transport pathway in a manner required for their mutual processing in response to glucocorticoids and establishes the importance of functional interactions with intracellular membranes for maturation of the cytoplasmic MMTV phosphoproteins.
Kain
S R
SR
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Platt
E J
EJ
Brown
K S
KS
Black
N
N
Firestone
G L
GL
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005938:Glucocorticoids / Q000494:pharmacology*; D006056:Golgi Apparatus / Q000187:drug effects; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000187:drug effects; D008114:Liver Neoplasms, Experimental / Q000378:metabolism* / Q000382:microbiology; D008324:Mammary Tumor Virus, Mouse / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D003517:Cyclopentanes; D005938:Glucocorticoids; D010750:Phosphoproteins; D014759:Viral Envelope Proteins; D020126:Brefeldin A