Vesicle-binding properties of wild-type and cysteine mutant forms of oil domain of apolipoprotein B

Previous studies demonstrated that structural perturbation of the oil domai n of apolipoprotein B (apoB) blocked the initiation of lipoprotein assembly , We explored the hypothesis that this domain may interact with the inner l eaflet of the endoplasmic reticulum membrane in a manner that may nucleate microsomal triglyceride transfer protein-dependent lipid sequestration. Apo B-17 (amino-terminal 17% of apoB), which contains most of the alpha (1) dom ain, was expressed stably in rat hepatoma cells and recovered from medium i n lipid-poor Form, On incubation with phospholipid vesicles composed of 1-m yristol-2-myristoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn- gylycero-3-phosphocholine, apoB-17 underwent vesicle binding and was recove red in the d < 1.25 g/ml gradient fraction. To determine whether vesicle bi nding is disrupted by the same structural perturbations that block lipoprot ein assembly in vivo, apoB-17 was subjected to partial and complete chemica l reduction. Although normally a soluble peptide, mild reduction of apoB-17 caused its precipitation, suggesting that hydrophobic, solvent-inaccessibl e domains within the or, domain of apoB are stabilized by intramolecular di sulfide bonds, In contrast to apoB-17 chemically reduced in vitro, forms of apoB-17 bearing pairwise cysteine-to-serine substitutions were recovered i n soluble form from transiently transfected COS-I cell extracts, Although i ndividual disruption of disulfide bond 2 or 4 in apoB-28 and apoB-50 was pr eviously shown to block lipoprotein assembly in vivo, these alterations had no impact on the ability of apoB-17 to bind to phospholipid vesicles in vi tro or on its capacity to form recombinant lipoprotein particles. These res ults suggest that while the vesicle/lipid-binding property of the rrl domai n may reflect an essential role required for the initiation of lipoprotein formation, some other aspect of oil domain Function is perturbed by disrupt ion of native disulfide bonds.