Transport of a lysosomally targeted Rous sarcoma virus envelope glycoprotein involves transient expression on the cell surface.
The details of intracellular transport pathways for glycosylated proteins remain incompletely described. We previously described a mutant Rous sarcoma virus envelope glycoprotein (gp), mu 26, with an altered membrane-spanning domain that was targeted to lysosomes after traversing the trans-Golgi. This mutant protein was not detectable on the cell surface by immunofluorescence, but its pathway for degradation remained unclear. To investigate this we have employed a second env mutation, S19, that results in a protein which is defective for normal cleavage/activation by intracellular enzymes, but remains susceptible to cleavage by extracellular proteases. Cleavage/activation of the double mutant by trypsin, which could only occur if it was exposed on the cell surface, was observed, indicating that the plasma membrane is an intermediate destination in the transport of this mutant protein. To substantiate these results, cells expressing the mu 26 glycoprotein were incubated with an antibody specific for the native protein in the presence of chloroquine. The specific accumulation of this antibody/gp complex in vesicles, as detected by internal immunofluorescence, confirmed the trypsin cleavage results. We conclude that this rapidly degraded mutant protein is transported from the trans-Golgi to the cell surface, where it is only transiently exposed, and then rapidly endocytosed and lysosomally degraded. The relevance of these results to the targeting of lysosomal proteins is discussed.
Johnston
P B
PB
Department of Microbiology, University of Alabama at Birmingham 35294.
Dong
J Y
JY
Hunter
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001358:Avian Sarcoma Viruses / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002738:Chloroquine / Q000494:pharmacology; D004705:Endocytosis; D015752:Genes, env; D006023:Glycoproteins / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D014759:Viral Envelope Proteins / Q000378:metabolism*
D000906:Antibodies; D006023:Glycoproteins; D014759:Viral Envelope Proteins; D002738:Chloroquine