Amino acid sequences within the 1 domain of human apolipoprotein B can mediate rapid intracellular degradation

Apolipoprotein B (apoB)-48 contains a region termed the 1 domain that is predicted to be composed of extensive amphipathic -strands. Analysis of truncated apoB variants revealed that sequences between the carboxyl termini of apoB-37 and apoB-42 governed the secretion efficiency and intracellular stability of apoB. Although apoB37, apoB-34, and apoB-29 were stable and secreted efficiently, apoB-42 and apoB-100 were secreted poorly and were degraded by an acetyl-leucyl-leucyl-norleucinal (ALLN)sensitive pathway. Amino acid sequence analysis suggested that a segment between the carboxyl termini of apoB-38 and apoB-42 was 63% homologous to fatty acid binding proteins (FABPs), which contain orthogonal -sheets. To test the hypothesis that sequences from the 1 domain are involved in apoB degradation, fusion proteins were created that contained apoB-29 linked to fragments derived from the 1 domain of apoB or to liver FABP. Fusion proteins containing the 1 domain segments apoB-34–42 or apoB-37–42 were degraded rapidly, whereas other fusion proteins were stable and secreted efficiently. Degradation was ALLN-sensitive, and the apoB-34–42 segment increased the association of the apoB protein with the cytosolic surface of the microsomal membrane. Our data suggest that the presence of specific sequences in the 1 domain of human apoB increases degradation by promoting the cytosolic exposure of the protein, although not all regions of the 1 domain are functionally equivalent.— Lapierre, L. R., D. L. Currie, Z. Yao, J. Wang, and R. S. McLeod. Amino acid sequences within the 1 domain of human apolipoprotein B can mediate rapid intracellular degradation. J. Lipid Res. 2004. 45: 366–377. Supplementary key words endoplasmic reticulum-associated degradation • proteasome • translocation Apolipoprotein (apo) B is a large and hydrophobic protein that forms the structural backbone for the assembly of triglyceride (TG)-rich lipoproteins (1, 2). Two forms of apoB are found in nature: the full-length apoB-100 (4,536 amino acids) and the truncated apoB-48 (2,152 amino acids), representing the amino-terminal 48% of apoB-100. Both forms are encoded by the same gene, and apoB-48 is the product of an mRNA-editing process (2–4) that generates an in-frame translation termination codon near the middle of the apoB transcript. Despite the difference in polypeptide length, both apoB-100 and apoB-48 contain sufficient sequence information for the assembly and secretion of large TG-rich lipoproteins, suggesting that the ability to recruit neutral lipids during lipoprotein assembly is encoded within the amino-terminal 48% of apoB-100. The structural elements within the apoB polypeptide that are responsible for lipid recruitment into a lipoprotein particle are poorly characterized. The amino-terminal 670 residues of apoB ( apoB-15) are disulfide-linked and form a globular domain that is homologous to the primitive lipid transport protein lipovitellin (5) and microsomal triglyceride transfer protein (MTP) (6). However, this region of apoB lacks the ability to recruit substantial quantities of neutral lipid (7). The remainder of the apoB polypeptide may form a “belt” that surrounds the lipoprotein (8), such that the length of the polypeptide determines the size of the nascent particle (9–11). Amphipathic -helix and -sheet (12) structures are thought to underlie the unique ability of apoB to assemble lipoproteins with a neutral lipid core (containing cholesteryl ester and TG), and amphipathic -structures have Abbreviations: ALLN, acetyl-leucyl-leucyl-norleucinal; apoB, apolipoprotein B; ER, endoplasmic reticulum; FABP, fatty acid binding protein; MTP, microsomal triglyceride transfer protein; PDI, protein disulfide isomerase; PNS, post-nuclear supernatant; PVDF, polyvinylidene difluoride; TG, triglyceride. 1 To whom correspondence should be addressed. e-mail: [email protected] Manuscript received 6 March 2003 and in revised form 13 August 2003. Published, JLR Papers in Press, October 27, 2003. DOI 10.1194/jlr.M300104-JLR200 by gest, on D ecem er 2, 2017 w w w .j.org D ow nladed fom