IDENTIFICATION OF CYSTEINE PAIRS WITHIN THE AMINO-TERMINAL 5-PERCENT OF APOLIPOPROTEIN-B ESSENTIAL FOR HEPATIC LIPOPROTEIN ASSEMBLY AND SECRETION

There is growing evidence that the amino-terminal globular domain of a polipoprotein B (apoB) is essential for lipoprotein particle formation in the hepatic endoplasmic reticulum, To identify the structural requ irements for its function in lipoprotein assembly, cysteine (Cys) pair s required to form the seven disulfide bonds within the amino-terminal 21% of apoB were replaced in groups or individually by serine. Substi tution of Cys pairs required for formation of disulfide bonds 1-3 or 4 -7 (numbered from amino to carboxyl terminus) completely blocked the s ecretion of apoB28 in transfected HepG2 cells. To identify the specifi c disulfide bonds required for secretion, Cys pairs were mutated indiv idually, Substitution of Cys pairs required for disulfide bonds 1, 3, 5, 6, or 7 had little or no impact on apoB28 secretion or buoyant dens ity. In contrast, individual substitution of Cys pair 2 (amino acid re sidues 51 and 70) or 4 (218 and 234) severely inhibited apoB28 secreti on and its capacity to undergo intracellular assembly with lipid. The same assembly and secretion defects were observed when these mutations were expressed as part of apoB50. These studies provide direct eviden ce that the ability of the internal lipophilic regions of apoB to enga ge in the recruitment and sequestration of lipid during translation is critically dependent upon a structural configuration contained within or affected by the amino terminal 5% of the protein.