Abstract
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.
Highlights
We have previously shown that glucocorticoids reg- Glucocorticoid hormones elicit their biological effects in a ulate the trafficking and processing of mouse mam- variety of target tissues by selectively modulating the tranmary tumor virus (MMTV) proteins in viral-infected scription of glucocorticoid-responsive genes [1,2,3,4,5]
To verify that brefeldin A (BFA) appropriately prevents the transport of MMTV glycoproteins in viral-infected M1.54 hepatoma cells, the localization of the cell surface viral glycoproteins in glucocorticoidtreated cells was examined by immunofluorescence microscopy
Monolayer cultures ofM1.54 cells were treated with either 1PM dexamethasone alone or incombination with 2.0 pg/ml BFA for 16 h and incubated with primary total antiMMTV antiserum and secondary fluorescein isothiocyanateconjugated anti-goat IgG.As shown in Fig. 1, cells treated with dexamethasone in the absence of BFA display a prominent immunofluorescence capping pattern indicative of cell
Summary
Digestion with Endoglycosidase H and N-Glycosidase F-For endoglycosidase H (endo H) digestions, the final Pansorbin pellets from immunoprecipitation of cell extracts were resuspended in 45 pl of 100 m M sodium citrate buffer, pH 5.5, containing 1%SDS, boiled for 3 min, and centrifuged in an Eppendorf microcentrifuge for 10 min. The supernatant fractions were collected and divided into 2O-pl aliquots, and 20 p1 of sodium citrate alone or 20 pl of sodium citrate containing 0.2 milliunits of endo H added to each sample. NGlycosidase F (endo F) digestions were performed by the same protocol, except Pansorbin pellets were resuspended in PBS containing. 1% SDS, andthe reaction mixture consisted of 20 pl of sample supernatant, 15 pl of PBS, 4 pl of n-octyl glucoside, in the presence or absence of 0.1 units of endo F.
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