Quantitative Analysis of ABCA1-dependent Compartmentalization and Trafficking of Apolipoprotein A-I

The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that 125I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I Δ(187–243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of 125I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of 125I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 °C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL. The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that 125I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I Δ(187–243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of 125I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of 125I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 °C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL. The molecular interaction of apoA-I with the cell membrane ABCA1 transporter has important implications in reverse cholesterol transport. It provides a mechanism whereby excess cholesterol is removed from peripheral cells that are unable to catabolize cholesterol, including macrophages in the vessel wall. This process plays a crucial role in both the formation and maintenance of HDL 3The abbreviations used are: HDL, high density lipoprotein; 22OH, 22(R)-hydroxycholesterol; 9CRA, 9-cis-retinoic acid; PAGGE, polyacrylamide nondenaturing gradient gel electrophoresis; ABCA1, ATP-binding cassette AI; apo, apolipoprotein; HCBS, high capacity binding site; ICCs, intracellular compartments; LpA-I, nascent apoA-I-containing particle; rLpA-I, recombinant LpA-I; PLC, phospholipase C; PC, phosphatidylcholine; PM, plasma membrane; TD, Tangier disease; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; Tf, transferrin; WT, wild type; SMase, sphingomyelinase; BHK, baby hamster kidney; CsA, cyclosporin A. levels in plasma and is believed to be one of the major mechanisms by which HDL protects against atherosclerotic cardiovascular disease (1Brewer Jr., H.B. Remaley A.T. Neufeld E.B. Basso F. Joyce C. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1755-1760Crossref PubMed Scopus (151) Google Scholar, 2Linsel-Nitschke P. Tall A.R. Nat. Rev. Drug Discov. 2005; 4: 193-205Crossref PubMed Scopus (417) Google Scholar). Despite a large body of information identifying HDL as a potent physiological protector against atherosclerotic cardiovascular disease, the fundamental mechanisms underlying the genesis of HDL at the cellular level remain complex and poorly understood. It is generally thought that the lipidation of apoA-I occurs inside the cell as a part of a retroendocytosis pathway. Of particular interest is the concept of Smith and co-workers (3Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar) that cellular cholesterol efflux involves endocytosis and resecretion of apoA-I. This concept falls in line with the idea that the ABCA1 transporter may play an important role in the apoA-I retroendocytosis pathway and is supported by previous studies documenting the following: 1) apoA-I colocalizes with ABCA1-containing endosomes (4Smith J.D. Waelde C. Horwitz A. Zheng P. J. Biol. PubMed Scopus Google and efflux is in the disease Y. C. A. A.R. Tall A.R. J. Biol. PubMed Scopus Google Scholar) and disease A.R. Tall A.R. J. Biol. PubMed Scopus Google Scholar). The and studies by Neufeld E.B. Remaley A.T. J. S. Jr., H.B. J. Biol. PubMed Scopus Google Scholar, E.B. Jr., A. J. Remaley A.T. S. Jr., H.B. J. Biol. 2004; PubMed Scopus Google Scholar) with a in cells have to important the mechanisms in the lipidation of apoA-I. ABCA1 was found to be in endosomes and and to between endosomes and the plasma membrane The of both apoA-I and ABCA1 in endosomes is thought to be important in efflux from intracellular It is apoA-I a membrane that is by ABCA1 A. 2004; PubMed Scopus Google Scholar, A. J. 2004; PubMed Scopus Google Scholar, C. P. S. J. Biol. 2004; PubMed Scopus Google Scholar) apoA-I/ABCA1 and be together for nascent HDL This information is to the of PM intracellular compartments cellular for the lipidation of apoA-I. is that a retroendocytosis pathway plays an important role in the formation of nascent HDL the the dynamics of apoA-I lipidation at cellular have we have used a quantitative assay cell surface biotinylation to the cellular compartmentalization and trafficking of apoA-I/ABCA1 in relation to the of nascent HDL we from and two with for at the ABCA1 and for as A. C. S. S. J. S. Jr., J. Nat. 1999; PubMed Scopus Google The for the study was and by the of the for and cells stably with an ABCA1 that is by the cells with and cells with the the ABCA1 by F. from the of of and and as J. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). cells from of the of and and in Dulbecco's modified Eagle's with and plasma apoA-I was in and against apoA-I was with by to a of apoA-I and used of wild apoA-I and in a and the and of have by J. F. P. PubMed Scopus Google Scholar). apoA-I was by cells stably ABCA1 with as by and co-workers J. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). with and 9-cis-retinoic for in the of of apoA-I for at 37 with of apoA-I. with PBS, and surface with for at °C. The biotinylation was for at by of the biotin and of with PBS, and of was to of and a at °C. The (PM) was with and for To the biotinylation of cell surface was cells with and with of 125I-apoA-I for at and biotinylation was as the of 125I-apoA-I associated with PM was from 125I-apoA-I to the We found that than of apoA-I at the cell surface was to of HDL apoA-I and the as by A. A. J. Biol. PubMed Google Scholar). of was particles by molecular to apoA-I complex formation was by with as we have S. S. J. J. 2005; PubMed Scopus Google Scholar). ABCA1 as J. Biol. 1999; PubMed Scopus Google Scholar). ABCA1 with a cleavable of biotin at for biotin was and cells with apoA-I at 37 for various to cellular cells with PBS, and biotin was a in which was to the cells for was by two with at °C. cells with apoA-I at assay and to used as for the of biotin than of ABCA1 was by and which to the plasma membrane in cells and used as for The of ABCA1 over and ABCA1 over To that of cleavable biotin the of ABCA1, cholesterol efflux We found that cell surface with cleavable biotin cholesterol efflux ABCA1 pathway. of from with ABCA1 mutations (Q597R and from Tangier disease to in and with and 9-cis-retinoic for in cells stably ABCA1 with as in the of 125I-apoA-I for at 37 °C. to was and the at 37 for was performed, and 125I-apoA-I dissociation from both plasma membrane and intracellular compartments was as of to the at the time from cells was by PAGGE, and the of apoA-I was by with as Jr., J. J. Biol. 2004; PubMed Scopus Google Scholar). with cells for in to cells to treatment with phosphatidylcholine-specific phospholipase for at 37 °C. cell surface biotinylation was as compared by as of a the cellular compartmentalization and trafficking of apoA-I in a cell we developed a quantitative assay cell surface This to the of apoA-I associated with the PM and ICCs as both the biotinylation of cell surface the of PM with ICCs in an To the biotinylation of cell surface was with 125I-apoA-I for at 37 and with of biotin 125I-apoA-I associated with PM ICCs was by the of biotin in in 125I-apoA-I association with the PM ICCs To the of the biotinylation a excess of apoA-I, of ABCA1 with and ABCA1 used as in the of excess apoA-I, the of with of an ABCA1 reduced the association of 125I-apoA-I with both the PM and ICCs. We have that the apoA-I is and to the cell surface Jr., J. J. Biol. 2004; PubMed Scopus Google Scholar). To the of the PM and ICCs both by and monitored by for the of cellular of the PM with ICCs was as by the of from ICCs, which is a for the PM. and found associated with ICCs that the biotinylation is and to the PM. of a of biotin and an of at used for biotinylation a of of of 125I-apoA-I with cells at 37 was to time for of apoA-I with cellular compartments. used binding at to determine the association of apoA-I with the PM, we evidence that at the association of apoA-I with ABCA1 at the PM in with previous studies Jr., J. J. Biol. 2004; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). the of physiological to study apoA-I/ABCA1 of with the PM and determine the association of apoA-I with the PM and ICCs, cells with with with of 125I-apoA-I for at 37 °C. to biotinylation was and 125I-apoA-I associated with the PM and ICCs was by in 125I-apoA-I exhibited association with the PM and ICCs in cells with 125I-apoA-I association with in cells 125I-apoA-I exhibited saturable association with both PM and ICCs in of for the association of apoA-I with cellular compartments revealed that the PM a 2-fold higher capacity to accommodate apoA-I as compared with ICCs in ABCA1 and fibroblasts. apoA-I exhibited for both the PM and ICCs This that of continuous to an excess of apoA-I, of apoA-I is found associated with the PM and the with the of apoA-I association and dissociation from the PM and ICCs in cells and cell cell for PM is compared with ICCs in for PM is compared with ICCs in for PM is compared with ICCs in for PM is compared with ICCs in fibroblasts. for PM is compared with ICCs in for PM is compared with ICCs in for PM is compared with ICCs in for PM is compared with ICCs in fibroblasts. in a a that is to specifically with the and to the cell surface J. PubMed Scopus Google Scholar) was used as a We found that of was found associated with the PM and with ICCs in of of cell of the C-terminal of and the of the with the PM and is that the C-terminal of apoA-I is important in the efflux pathway. previous studies have that apoA-I deletion of the C-terminal both reduced cell surface binding and the to efflux A. 2004; PubMed Scopus Google Scholar, C. P. S. J. Biol. 2004; PubMed Scopus Google Scholar, J. F. P. PubMed Scopus Google Scholar). we and have that association with apoA-I reduced to with ABCA1 Jr., J. J. Biol. 2004; PubMed Scopus Google Scholar, S. J. J. 2005; PubMed Scopus Google Scholar, Jr., J. J. 2004; PubMed Scopus Google Scholar, Tall A.R. J. Biol. PubMed Scopus Google Scholar). We the role of the C-terminal of apoA-I and the lipidation of apoA-I the association with cellular compartments. in and apoA-I Δ(187–243) and exhibited reduced cell association and association with both the PM and ICCs as compared with apoA-I. that the C-terminal of apoA-I is important for the compartmentalization of apoA-I between the PM and ICCs. a of apoA-I for Overexpressing ABCA1 the of between the PM and is that ABCA1 in endocytic plays a role in cellular efflux (4Smith J.D. Waelde C. Horwitz A. Zheng P. J. Biol. PubMed Scopus Google Scholar, E.B. Jr., A. J. Remaley A.T. S. Jr., H.B. J. Biol. 2004; PubMed Scopus Google Scholar, C. A. PubMed Scopus Google Scholar, Tall A.R. J. Biol. 2005; PubMed Scopus Google Scholar). To the role of ABCA1 in the cellular compartmentalization of apoA-I, levels of ABCA1 in cells a ABCA1 in cells ABCA1 cells with and for levels of cellular cholesterol efflux was increased and and ABCA1 by was by 125I-apoA-I association to both the PM and ICCs of with increased the association of 125I-apoA-I with both the PM and ICCs that of ABCA1 is required for the association of apoA-I with both the PM and ICCs. The for the of with that ABCA1 is required for the compartmentalization of apoA-I, we the of ABCA1 trafficking apoA-I association with the PM and ICCs. with cyclosporin which are to specifically cholesterol efflux to apoA-I Smith J.D. Arterioscler. Thromb. Vasc. Biol. 2004; 24: PubMed Scopus Google Scholar, S. J. Biol. 2004; PubMed Scopus Google Scholar). is a potent of ABCA1 to recycling of ABCA1 from the PM to in the of HDL and of ABCA1 by Smith J.D. Arterioscler. Thromb. Vasc. Biol. 2004; 24: PubMed Scopus Google Scholar). and co-workers F. F. Arterioscler. Thromb. Vasc. Biol. 2004; 24: PubMed Scopus Google Scholar) that cholesterol efflux by trafficking of ABCA1 to the PM. and co-workers S. J. Biol. 2004; PubMed Scopus Google Scholar) of the trafficking of is to the interaction of ABCA1 with apoA-I and to of ABCA1 S. J. Biol. 2004; PubMed Scopus Google Scholar). treatment of cells with reduced the of apoA-I and cholesterol efflux This was of a in the association of 125I-apoA-I with ICCs the PM both and a in ABCA1 association with the PM Despite in we a in apoA-I. may be to a of ABCA1 for apoA-I by and that of apoA-I and ABCA1 trafficking has a the apoA-I endocytotic pathway and the apoA-I lipidation ABCA1 that of ABCA1 endocytic trafficking apoA-I association with ICCs and the was as to ABCA1 at the cell surface is together with apoA-I the lipidation To the of we a cleavable biotinylation PM are with a cleavable biotin at and are to 37 to cellular with 125I-apoA-I for various of cells with a which PM that have the biotin an of the and level of cells at and to a used as for the of biotin by as in a of ABCA1 was the of the assay in the of apoA-I in the of ABCA1 a in the of apoA-I, ABCA1 exhibited in the of apoA-I in cells This was in to a PM level of the The of ABCA1 over is in The in ABCA1 was to an in ABCA1 the of ABCA1 It is that the the of the intracellular ABCA1 which the of ABCA1 endocytosis and recycling to the PM. with (Q597R and C1477R). The of ABCA1 over in increased of ABCA1 ABCA1 associated with in the of apoA-I. experiment of ABCA1 in and is and we found that ABCA1 was impaired in the ABCA1 associated with that apoA-I induces the of We are the of endocytosis and recycling of ABCA1 and to the formation of nascent HDL. of from the PM and the of the PM and ICCs to the lipidation of apoA-I, the dissociation (re-secretion) of apoA-I from compartments was monitored over a 6-h period. was for at 37 with fibroblasts. to cells with for various in and the of 125I-apoA-I dissociation from the PM and from ICCs revealed that the time required for 50% dissociation of 125I-apoA-I from the PM was 4-fold slower than that of ICCs at 37 in both cell the that from the cell as 125I-apoA-I in the over a 6-h of 125I-apoA-I from the PM and ICCs over the 6-h was at of the to the and in cell was by that endocytosis of apoA-I, with previous Jr., J. J. Biol. 2004; PubMed Scopus Google Scholar) by in which a of apoA-I from both the PM and ICCs was the of molecular of the 125I-apoA-I by revealed the of nascent apoA-I-containing particles with of as we have S. S. J. J. 2005; PubMed Scopus Google Scholar, Jr., J. J. Biol. 2004; PubMed Scopus Google Scholar). cells unable to particles The of particles by nascent particles of apoA-I as we and have Jr., J. J. Biol. 2004; PubMed Scopus Google Scholar, C. P. S. J. Biol. PubMed Scopus Google Scholar) that apoA-I was rapidly from the ICCs dissociation from the PM, and apoA-I was from both compartments as nascent apoA-I-containing of from the PM by we and have a PM with a high binding capacity for apoA-I J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, C. S. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar). of we that apoA-I be by treatment with phosphatidylcholine-specific phospholipase in treatment of with increased 125I-apoA-I dissociation from the PM. the of apoA-I from ICCs was by treatment reduced by the of apoA-I associated with the plasma membrane treatment the of apoA-I associated with ICCs. To removed by the cells with and the by and and treatment than of and of membrane and cellular phospholipase treatment monitored by in was by treatment compared with cells that the association of apoA-I with the PM be by and that a large of apoA-I is J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, C. S. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar). the of cellular apoA-I and is for nascent HDL genesis occurs at the cellular the HDL retroendocytosis was by and co-workers U. J. 4: PubMed Scopus Google Scholar) and physiological remain studies by and Smith (3Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar) and Neufeld E.B. Remaley A.T. J. S. Jr., H.B. J. Biol. PubMed Scopus Google Scholar, E.B. Jr., A. J. Remaley A.T. S. Jr., H.B. J. Biol. 2004; PubMed Scopus Google Scholar) have a in which the endocytotic pathway plays an important role in the formation of HDL This is with the of Tall and co-workers Tall A.R. J. Biol. 2005; PubMed Scopus Google Scholar) showing that deletion of the to of ABCA1 and reduced cholesterol efflux from cholesterol a by and co-workers C. A. PubMed Scopus Google Scholar) that ABCA1 apoA-I in studies the concept that the and trafficking of ABCA1 is important in the intracellular lipidation of apoA-I. the of two cellular compartments for the of nascent HDL We evidence that of continuous to an excess of apoA-I, the PM exhibited a 2-fold higher capacity to accommodate apoA-I as compared with ICCs We that the PM is to accommodate apoA-I of the of a binding as by J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar) as as and co-workers C. S. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar). Despite the that of apoA-I was found ICCs, 125I-apoA-I was from ICCs dissociation from the PM and both apoA-I from ICCs from the PM was as nascent HDL the kinetic that ICCs a for the lipidation of apoA-I, in with previous (3Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar, E.B. Jr., A. J. Remaley A.T. S. Jr., H.B. J. Biol. 2004; PubMed Scopus Google the quantitative suggests an important role of the PM to apoA-I and the idea that the PM an lipidation This is with previous that of the the formation of nascent HDL J. J. PubMed Scopus Google Scholar). It is that the PM as an apoA-I apoA-I to be for interaction with the endocytotic pathway. The of the between the of apoA-I dissociation from the PM and from ICCs is may be to the of apoA-I by PM with the data showing that treatment increased the dissociation of apoA-I from the PM be that apoA-I from ICCs with the PM, showing that apoA-I from both the PM and ICCs are together with the finding that exhibited association with the PM and ICCs a The for apoA-I to with the PM and ICCs are as study we evidence that deletion of the C-terminal of apoA-I, reduced the compartmentalization of apoA-I between the PM and ICCs with previous report documenting that apoA-I Δ(187–243) exhibited reduced binding to both ABCA1 and the J. J. PubMed Scopus Google Scholar). This is in with a previous study by and co-workers A. 2004; PubMed Scopus Google Scholar) showing that of apoA-I and to ABCA1 higher for compared with apoA-I. It is that the C-terminal are important for cellular efflux A. 2004; PubMed Scopus Google Scholar, C. P. S. J. Biol. 2004; PubMed Scopus Google Scholar). we have that apoA-I with to nascent particles compared with apoA-I J. PubMed Scopus Google Scholar). that the C-terminal of apoA-I is required for the formation of a complex with ABCA1 that to the compartmentalization of apoA-I between the PM and ICCs. of compartmentalization by the C-terminal deletion be to an of apoA-I to to as the the PM J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, C. S. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar) to an of the apoA-I endocytotic pathway. This study the concept that the association of apoA-I with the PM and ICCs is the formation of a high complex between apoA-I and ABCA1, which the compartmentalization and trafficking of apoA-I. We that apoA-I binding to ABCA1 by by promoted the association of apoA-I to both the PM and ICCs and of the high complex formation by deletion of the C-terminal by lipidation of apoA-I impaired the association of apoA-I with both the PM and ICCs. that the interaction between apoA-I and ABCA1 plays a role in the cellular compartmentalization and trafficking of apoA-I, the formation of nascent HDL particles at compartments. is that capacity apoA-I/ABCA1 interaction a and ABCA1, as by and co-workers C. S. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar). the mechanisms ABCA1 and to the compartmentalization and trafficking of apoA-I remain previous studies by Tall and co-workers Tall A.R. J. Biol. PubMed Scopus Google Scholar) have that apoA-I increased cell surface ABCA1 by by we that apoA-I induces continuous of We the ABCA1 transporter at the complex rapidly to of cell surface ABCA1 and of a large of apoA-I lipidation This is supported by the 1) specifically mediates the continuous endocytic recycling of ABCA1, and process is impaired in cells The of 125I-apoA-I from ICCs was than dissociation from the PM in apoA-I/ABCA1 trafficking impaired the lipidation process showing that of the high capacity binding impaired HDL formation J. J. PubMed Scopus Google Scholar) that a complex mechanism for the lipidation of apoA-I compartments. It is that of continuous to an excess of apoA-I the of apoA-I compartments the of two of endocytosis from the PM and from ICCs. This is with the concept that the trafficking of ABCA1 is for the lipidation of apoA-I intracellular for lipidation the high capacity binding at the PM. It is that binding the of cellular and endocytosis in of apoA-I may have important implications in the of ABCA1, with previous study Jr., J. J. Biol. 2004; PubMed Scopus Google Scholar) which that treatment of hamster cells overexpressing ABCA1 with apoA-I and ABCA1 This is in with the finding of and Rev. 2005; PubMed Scopus Google Scholar) that ABCA1 be by including the pathway. apoA-I to both the and of ABCA1 found in both the PM and compartments as we have the of apoA-I the of ABCA1 Jr., J. J. Biol. 2004; PubMed Scopus Google Scholar). cellular the of apoA-I are poorly a study by and co-workers Scholar) that of cells overexpressing ABCA1 with apoA-I for at 37 apoA-I is for we evidence that of apoA-I is increased 2-fold as compared with with apoA-I for at 37 as by and co-workers A. J. 2004; PubMed Scopus Google Scholar) that in the complex has a of than and that 50% of the apoA-I is we S. S. J. J. 2005; PubMed Scopus Google Scholar) that in cells and formation of nascent particles occurs at 37 and 1) that the of dissociation from the ICCs is with a of a large of lipidation have of in have reduced the of the the of excess cellular cholesterol is that apoA-I be for This is with finding that of by treatment of cells with for at 37 by with 125I-apoA-I for at 37 a of 125I-apoA-I compared with cells are required to cellular the of apoA-I. major in the concept of reverse cholesterol from the of and co-workers F. Remaley A. J. Neufeld E.B. J. S. Jr., H.B. J. PubMed Scopus Google that the is a major of plasma This concept is supported by a study by and co-workers A. Smith J. 2005; PubMed Scopus Google Scholar) showing that of ABCA1 in reduced the level of HDL. It be of interest to determine mechanisms that we for the lipidation of apoA-I in and and co-workers J. J. Biol. 2005; PubMed Scopus Google Scholar) have that apoA-I in an in the that is by in has that apoA-I cholesterol in the and that the major occurs at the cell surface of with showing the capacity of the PM to with apoA-I the kinetic data that the endocytotic pathway plays a central role in the formation of nascent HDL, in with of nascent HDL apoA-I to ICCs in cholesterol may have important implications in atherosclerotic cardiovascular This is supported by the finding of and co-workers Y. A.R. Nat. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar) that the is the of in This study has for a quantitative to be between the of apoA-I from the PM and ICCs. we evidence that the between apoA-I and ABCA1 endocytic trafficking plays a physiological role in the lipidation a lipidation for nascent HDL as in The interaction of apoA-I with ABCA1 at the cell surface a apoA-I high capacity binding at the PM J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, C. S. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar) apoA-I and is from the PM as nascent HDL particles lipidation involves endocytosis of both apoA-I and ABCA1 by dissociation and of apoA-I to ICCs. ABCA1 is to of cell surface from intracellular and is rapidly from the cell as nascent HDL particles lipidation we to that was to to a the lipidation of apoA-I at cellular compartments. in is a of and previous the J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar) and of the in the The mechanisms underlying HDL genesis at the cellular level which are of a kinetic as is a for a complex intracellular trafficking pathway for apoA-I/ABCA1 that plays an important role in cellular cholesterol and the genesis of nascent HDL We F. and for cells overexpressing ABCA1 and for apoA-I

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