Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment.
The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0-10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.
Persson
R
R
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Ahlström
E
E
Fries
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000418:Albumins / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D001386:Azides / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006242:Haptoglobins / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D011919:Rats, Inbred Strains; D019810:Sodium Azide
D000418:Albumins; D001386:Azides; D006242:Haptoglobins; D000255:Adenosine Triphosphate; D019810:Sodium Azide