Reconstituting initial events during the assembly of apolipoprotein B-containing lipoproteins in a cell-free system.
The synthesis of apolipoprotein B (apoB) dictates the formation of chylomicrons and very low-density lipoproteins, two major lipoprotein precursors in the human plasma. Despite its biological significance, the mechanism of the assembly of these apoB-containing lipoproteins remains elusive. An essential obstacle is the lack of systems that allow fine dissection of key components during assembly, including nascent apoB peptide, lipids in defined forms, chaperones, and microsomal triglyceride transfer protein (MTP). In this study, we used a prokaryotic cell-free expression system to reconstitute early events in the assembly of apoB-containing lipoprotein that involve the N-terminal domains of apoB. Our study shows that N-terminal domains larger than 20.5% of apoB (B20.5) have an intrinsic ability to remodel vesicular phospholipid bilayers into discrete protein-lipid complexes. The presence of appropriate lipid substrates during apoB translation plays a pivotal role for successful lipid recruitment, and similar lipid recruitment fails to occur if the lipids are added posttranslationally. Cotranslational presence of MTP can dramatically promote the folding of B6.4-20.5 and B6.4-22. Furthermore, apoB translated in the presence of MTP retains its phospholipid recruitment capability posttranslationally. Our data suggest that during the synthesis of apoB, the N-terminal domain has a short window for intrinsic phospholipid recruitment, the time frame of which is predetermined by the environment where apoB synthesis occurs. The presence of MTP prolongs this window of time by acting as a chaperone. The absence of either proper lipid substrate or MTP may result in the improper folding of apoB and, consequently, its degradation.
Jiang
Z Gordon
ZG
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA.
Liu
Yuhang
Y
Hussain
M Mahmood
MM
Atkinson
David
D
McKnight
C James
CJ
D001055:Apolipoproteins B / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002352:Carrier Proteins / Q000378:metabolism; D002474:Cell-Free System; D050356:Lipid Metabolism; D008958:Models, Molecular; D014176:Protein Biosynthesis; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D001055:Apolipoproteins B; D002352:Carrier Proteins; C106092:microsomal triglyceride transfer protein