ABCA1-mediated lipid efflux to lipid poor apoA-I results in the gradual lipidation of apoA-I. This leads to the formation of discoidal HDL which are subsequently converted to spherical HDL by the action of LCAT. We have investigated the effect of point mutations and deletions in the carboxyterminal region of apoA-I on the biogenesis of HDL using adenovirus-mediated gene transfer in apoA-I deficient mice. It was found that the plasma HDL levels were greatly reduced in mice expressing the carboxy-terminal deletion mutants apoA-I [Δ(185-243)] and apoA-I [Δ(220-243)], shown previously to diminish the ABCA1-mediated lipid efflux. The HDL levels were normal in mice expressing the WT apoA-I, the apoA-I[Δ(232-243)] deletion mutant or the apoA-I[E191A/ H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid efflux. Electron microscopy and two-dimensional gel electrophoresis showed that the apoA-I [Δ(185-243)] and apoA-I[Δ(220-243)] mutants formed mainly preβ-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I [Δ(232-243)] and apoA-I[E191A/H193A/K195A] formed spherical α-HDL particles. The findings establish that a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of α-HDL, but allow the synthesis of preβ-HDL particles in vivo, b) the aminoterminal segment 1-184 of apoA-I can promote synthesis of preβ-HDL type particles in an ABCA1-independent process and c) the charged residues in the 191-195 region of apoA-I do not influence the biogenesis of HDL.Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL), and plays an essential role in the biogenesis, structure, function and plasma concentration of HDL (1-5). ApoA-I contains 22-and 11-amino acid repeats (6;7) which based on earlier x-ray crystallography (8) and computer modeling (7) are organized in amphipathic α-helices. Most recently the lipid-free apoA-I has been crystallized in salt buffers containing 500 μM Cr(III)-Tris-acetylacetonate (Cr-acac3) (9). Under the conditions of crystallization, the protein consists of a four helix amino-terminal bundle and two carboxy-terminal helices.The biogenesis and catabolism of HDL can be considered as a complex pathway that involves several proteins (5). In the early steps of this pathway, apoA-I is secreted mostly lipid-free by Address correspondence to: Angeliki Chroni, Institute of Biology, National Center for Scientific Research "Demokritos", Agia Paraskevi, Athens 15310, Greece; Tel. +30 210 6503626; Fax. +30 210 6511767; Email: achroni@bio.demokritos.gr. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 3. Published in final edited form as:Biochemistry. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the liver and acquires phospholipid and cholesterol via its interactions with the ATP-binding cassette A1 (ABCA1) lipid transporter (2;10;11). Through a series of intermediate steps that are poorly understood, apoA-I is gradually lipidat...