The distribution of apolipoprotein B in endoplasmic reticulum and Golgi subfractions of rabbit liver

Abstract

The liver is the site of synthesis and secretion of very-lowdensity lipoproteins (VLDL), the vehicle for the transport of endogenous triacylglycerol (TAG) and the precursor of lowdensity lipoproteins (LDL). Elevated plasma levels of VLDL and LDL are recognized risk factors in the development of atherosclerosis. The production of VLDL by the liver is considered to be the most appropriate therapeutic target for the reduction of circulating lipoprotein levels. An understanding of the molecular and topographical events in the assembly of VLDL is thus of fundamental importance. Apolipoprotein B (apo B) is the major non-exchangeable protein of VLDL, and is essential for their secretion. Studies in HEP-G2 cells have indicated that apo B is synthesized by membrane-bound polysomes [I]. Newly synthesized apo B remains associated with the endoplasmic reticulum (ER) membrane for a period of time before being transferred to the cisternal space [2]. Consistent with this, we have previously shown that apo B does not associate with TAG-richVLDL precursors in the ER cisternae, and that the main site of packaging of VLDL lipid and protein is the Golgi cisternae [3]. To examine the intracellular events in apo B synthesis and translocation in more detail, we have determined the intracellular location and amount of apo B within the secretory pathway. The intracellular location of apo B was determined by SDS/PAGE and immunoblotting of subcellular fractions. ER and Golgi fractions were prepared from rabbit liver, and separated into membrane and cisternal content fractions by sodium carbonate treatment 14, 51. Fractions were delipidated and separated on 3-20% (w/v) polyacrylamidegradient gels [6], and then electroblotted on to nitrocellulose membrane [7]. Apo B- 100, the only apparent species of apo B secreted by rabbit liver, was detected by immunoblotting using sheep anti-rabbit apo B (primary antibody), and biotinylated anti-sheep IgG (secondary antibody). The secondary antibody was detected colorimetrically by the highly sensitive Vector ABC-alkaline phosphatase system. Apo €3- 100 was detectable in both ER and Golgi fractions (Fig. 1 ). When ER was separated into membranes and cisternal contents, apo B-100 was found in both fractions at apparently similar levels. When Golgi was separated into membranes and cisternal contents, although apo B- 100 could be detectcd in membranes, most of the protein appeared to be located in the contents. Apo €3- 100 in subcellular fractions was quantified by radioimmunoassay. LDL were isolated from rabbit plasma (d 1 .O 19-d 1.063). and radiolabclled with lz51 [ 8, 01. Apo B-I00 was the only protein detectable in LDL by SDS/ PAGE. and was found to be 'z51-labelled. Using a fixed concentration of labelled LDL with varying concentrations of