Expression of mouse mammary tumor virus glycoprotein truncations defines roles for the transmembrane domain and ectodomain hydrophobic region in constitutive exocytic trafficking and proteolytic processing.
A mutational analysis was used to identify structural domains that are important for exocytic transport and proteolytic cleavage of the mouse mammary tumor virus (MMTV) glycoprotein, which is expressed as a multidomain polyprotein. Rat HTC hepatoma cells were transfected with the MMTV glycoprotein gene driven by the constitutive Rous sarcoma virus promoter, with mutant genes encoding a series of polypeptide truncations or with a defective MMTV provirus containing a premature termination codon in the viral glycoprotein gene. Efficient proteolytic maturation and transport of MMTV glycoproteins to the cell surface or extracellular environment required the presence of the transmembrane domain but not the cytoplasmic tail. Two stable truncations retaining the hydrophobic region of the ectodomain in the absence of the transmembrane domain and cytoplasmic tail (trgp67 and trgp58) remained in endoglycosidase H sensitive and uncleaved forms. One of these truncations, trgp58, appeared to be tightly associated with intracellular membranes and strongly bound by heavy chain binding protein, whereas the other truncation, trgp67, was a soluble component of the lumen and persists intracellularly by a heavy chain binding protein-independent pathway. The truncated MMTV glycoprotein additionally lacking the hydrophobic region of the ectodomain was efficiently secreted. Taken together, our results demonstrate that the hydrophobic transmembrane domain of the MMTV glycoprotein is required for proper transport and proteolytic processing, whereas, in the absence of the transmembrane domain, the presence of a hydrophobic region of the ectodomain correlated with retention at an early step in the exocytic pathway.
Platt
E J
EJ
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Firestone
G L
GL
D015152:Blotting, Northern; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D011533:Proviruses / Q000378:metabolism; D012367:RNA, Viral / Q000032:analysis; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D012367:RNA, Viral