Abstract
Pulse-chase studies combined with subcellular fractionation indicated that LpB 100 (i.e. the apoprotein B (apoB) 100 containing lipoproteins) was released to the lumen of the secretory pathway in a subcellular fraction enriched in smooth vesicles, and referred to as SMF (the smooth membrane fraction). The migration of SMF during gradient ultracentrifugation as well as kinetic studies indicated that the fraction was derived from a pre-Golgi compartment, probably the smooth endoplasmic reticulum (ER). Only small amounts of LpB 100 could be detected during these pulse-chase experiments in the subcellular fractions derived from the rough endoplasmatic reticulum (RER). SMF contained the major amount of the diacylglycerol acyltransferase activity present in the ER, while the major amount of membrane bound apoB 100 was present in the RER. Pulse-chase studies of the intracellular transfer of apoB 100 demonstrated the formation of a large membrane-bound preassembly pool in the ER, while no significant amount of apoB 100 radioactivity was present in the membrane of the Golgi apparatus. The maximal radioactivity of LpB 100, recovered from the ER or the Golgi lumen, was small compared with the radioactivity recovered from the ER membrane, indicating that the assembled LpB 100 rapidly leaves the cells. This in turn indicates that the rate-limiting step in the secretion of apoB 100 was the transfer of the protein from the ER membrane to the LpB 100 in the lumen. A portion of the intracellular pool of apoB 100 was not secreted but underwent posttranslational degradation.
Highlights
Bondjers, of Goteborg, Olov Wiklund, Pulse-chase studies combined with subcellular fractionation indicated that LpB 100 (i.e. the apoprotein B 100 containing lipoproteins) was released to the lumen of the secretory pathway in a subcellular fraction enriched in smooth vesicles, and referred to as SMF
Sucrose gradient ultracentrifugation of the d < 1.065 g/ml supernatant recovered from the secretory pathway of the cell, showed the presence of only Fraction II, i.e. the major apoB lOOcontaining lipoprotein secreted into the medium
The size of LpB 100 recovered after 15 min of pulse and 15 min of chase from the endonlasmic reticulum (ER) fractions was very similar to that of the major apoB 100 lipoprotein secreted into the medium (Fig. 6B)
Summary
Materials-Eagle’s minimal essential medium was from Flow Laboratories (Irwine, United Kingdom) while Eagle’s minimal essential medium without methionine was from GIBCO. Cq-Antitrypsin was recovered from each fraction by immunoprecipitation and divided into two portions, one of which was incubated with Endo-H as described in Ref. 15 Both samples were analyzed by SDS-polyacrylamide gel electrophoresis, followed by autoradiography. Sucrose gradient ultracentrifugation of the d < 1.065 g/ml supernatant recovered from the secretory pathway of the cell, showed the presence of only Fraction II, i.e. the major apoB lOOcontaining lipoprotein secreted into the medium (cf Fig. 8 and Ref. 1). A dilution series was created for the labeled culture medium (%, l/z, %, %a, %o, and l/loo) in unlabeled culture medium (Fig. 1C) and apoB 100 was recovered from each sample as described above and the radioactivity counted. The results (Fig. 1C) showed a linear relation between the recovered apoB 100 radioactivity and the amount of labeled medium subjected to immunoprecipitation. Iktermination of Lipid-phosphorus-Lipids were extracted as described in Ref. 21, and the lipid-phosphorus was determined by the method described in Ref. 22
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