TY - JOUR
T1 - Transport of a lysosomally targeted Rous sarcoma virus envelope glycoprotein involvestransient expression on the cell surface
AU - Johnston, Patrick B.
AU - Doing, Jian Yun
AU - Hunter, Eric
N1 - Funding Information:
We are appreciative to James Collawn for insightful discussion. We appreciate technical help by Sally K. Weldon. For critical reviews of this manuscript, we thank David Einfeld and Robert Weldon. This work was supported by Public Health Service Grant CA-29884 from the National Institutes of Health. Patrick Johnston is supported by a scholarship from the Life and Health Insurance Medical Research Fund. The monoclonal antibody 1B5 was a gift from Mark Marsh at the MRC Laboratory for Molecular Cell Biology, University College, London. The monoctonal antibody H4B4 was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biology, University of Iowa, Iowa City, IA, under Contract NO1-HD-2-3144 from the NICHD.
PY - 1995
Y1 - 1995
N2 - The details of intracellular transport pathways for glycosylated proteins remain incompletely described. We previouslydescribed a mutant Rous sarcoma virus envelope glycoprotein (gp), μ26, with an altered membrane-spanning domain that was targeted to Iysosomes 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 μ26 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.
AB - The details of intracellular transport pathways for glycosylated proteins remain incompletely described. We previouslydescribed a mutant Rous sarcoma virus envelope glycoprotein (gp), μ26, with an altered membrane-spanning domain that was targeted to Iysosomes 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 μ26 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.
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U2 - 10.1016/S0042-6822(95)80050-6
DO - 10.1016/S0042-6822(95)80050-6
M3 - Article
C2 - 7831790
AN - SCOPUS:0028854178
SN - 0042-6822
VL - 206
SP - 353
EP - 361
JO - Virology
JF - Virology
IS - 1
M1 - 95800506
ER -