A growth hormone-vesicular stomatitis virus G hybrid protein is rapidly degraded in lysosomes following transport to the cell surface.
We have expressed the hybrid protein, GHG3, in baby hamster kidney cells to study protein turnover. GHG3 contains the cytoplasmic and transmembrane domains of vesicular stomatitis virus G protein linked to the C-terminus rat growth hormone. Turnover of GHG3 was prevented by lysosomal inhibitors (leupeptin, chloroquine, primaquine or monensin), while the accumulated GHG3 was localized to intracellular vesicles, results indicating that degradation occurred in lysosomes. The kinetics of degradation at 34 degrees C were determined in pulse-chase studies of metabolically labeled cells. After a lag period of 1 h, degradation was rapid (t1/2 = 1.25 h). The fate of GHG3 during the lag period was determined by immunofluorescence. We detected GHG3 on the cell surface when growth hormone antiserum was added to the growth medium 90 min prior to fixation and staining. No staining was observed if protein synthesis was inhibited with cycloheximide 90 min prior to the addition of growth hormone antiserum, a result indicating that GHG3 was rapidly removed from the cell surface. Unless the cells were pretreated with cycloheximide, antiserum was also detected in intracellular vesicles, which showed that GHG3 was endocytosed. These data indicate that a pool of GHG3 is transported rapidly to the cell surface, endocytosed and with little or no recycling directed to lysosomes for degradation.
Rizzolo
L J
LJ
Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322.
D000818:Animals; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D006224:Cricetinae; D004705:Endocytosis; D013006:Growth Hormone / Q000378:metabolism*; D007668:Kidney / Q000166:cytology* / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D007976:Leupeptins; D008565:Membrane Proteins; D014764:Viral Proteins; D002738:Chloroquine; D013006:Growth Hormone; D008985:Monensin; C032854:leupeptin