ABCMdb

PMID: 14617740

Fitzgerald ML, Morris AL, Chroni A, Mendez AJ, Zannis VI, Freeman MW
ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux.
J Lipid Res. 2004 Feb;45(2):287-94. Epub 2003 Nov 16., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCA1 p.Trp590Ser A mutant form of ABCA1 (W590S) that avidly binds apoA-I but fails to promote cholesterol efflux released apoA-I with similar kinetics but without transfer of cholesterol to apoA-I. Login to comment 7 ABCA1 p.Trp590Ser A mutant form of ABCA1 (W590S) that avidly binds apoA-I but fails to promote cholesterol efflux released apoA-I with similar kinetics but without transfer of cholesterol to apoA-I. Login to comment 126 ABCA1 p.Trp590Ser Previously, we had demonstrated that a mutant form of ABCA1, containing a missense mutation resulting in the substitution of serine for tryptophan at amino acid position 590, produced a transporter that had greater apoA-I cross-linking activity than wild-type ABCA1. Login to comment 127 ABCA1 p.Trp590Ser Previously, we had demonstrated that a mutant form of ABCA1, containing a missense mutation resulting in the substitution of serine for tryptophan at amino acid position 590, produced a transporter that had greater apoA-I cross-linking activity than wild-type ABCA1. Login to comment 128 ABCA1 p.Trp590Ser This mutant (W590S) retains the temperature dependence of cross-linking we previously demonstrated for wild-type ABCA1 (Fig. 4A), suggesting that its binding interaction Fig. 2. Login to comment 129 ABCA1 p.Trp590Ser This mutant (W590S) retains the temperature dependence of cross-linking we previously demonstrated for wild-type ABCA1 (Fig. 4A), suggesting that its binding interaction Fig. 2. Login to comment 140 ABCA1 p.Trp590Ser Thus, the dissociation of apoA-I from both wild-type ABCA1 and ABCA1(W590S) was tested. Login to comment 141 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Thus, the dissociation of apoA-I from both wild-type ABCA1 and ABCA1(W590S) was tested. Login to comment 142 ABCA1 p.Trp590Ser Cells expressing ABCA1, the W590S mutant, or no ABCA1 (mock) were exposed to 125I-apoA-I at 37ЊC for 1 h. Login to comment 146 ABCA1 p.Trp590Ser Fig. 4B (top panels) shows the amount of 125I-apoA-I associated with wild-type ABCA1 or the W590S mutant as assessed by the cross-linking assay. Login to comment 147 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Fig. 4B (top panels) shows the amount of 125I-apoA-I associated with wild-type ABCA1 or the W590S mutant as assessed by the cross-linking assay. Login to comment 148 ABCA1 p.Trp590Ser Although the W590S mutant was found to be associated with substantially more radiolabeled apoA-I at time zero, its dissociation rate from the apolipoprotein (half-life of 30.1 min) did not differ markedly from that measured for the wild-type transporter (half-life of 24.6 min) (Fig. 4B, graph). Login to comment 152 ABCA1 p.Trp590Ser Cells expressing the W590S mutant also released the apoA-I back into the medium at a similar rate. Login to comment 153 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Cells expressing the W590S mutant also released the apoA-I back into the medium at a similar rate. Login to comment 154 ABCA1 p.Trp590Ser These results, along with the measurements of the kinetics of dissociation described above, indicate that the cholesterol efflux defect in the W590S mutant cannot be accounted for by either a failure of the apoA-I/ABCA1 complex to dissociate or an ectopic release of apoA-I. Login to comment 155 ABCA1 p.Trp590Ser The apoA-I released from the cells expressing the W590S mutant migrated at the expected molecular weight of lipid-depleted, monomeric apoA-I (Fig. 4D). Login to comment 156 ABCA1 p.Trp590Ser The apoA-I released from the cells expressing the W590S mutant migrated at the expected molecular weight of lipid-depleted, monomeric apoA-I (Fig. 4D). Login to comment 169 ABCA1 p.Trp590Ser IP, immunoprecipitant. wild-type ABCA1 had acquired lipid, whereas the apoA-I released from cells expressing the W590S mutant had not. Login to comment 170 ABCA1 p.Trp590Ser IP, immunoprecipitant. wild-type ABCA1 had acquired lipid, whereas the apoA-I released from cells expressing the W590S mutant had not. Login to comment 172 ABCA1 p.Trp590Ser Cells that were either mock-transfected or that expressed the W590S mutant had similarly low levels of [14C]cholesterol associated with apoA-I in the medium (Fig. 4D, graph). Login to comment 173 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser Cells that were either mock-transfected or that expressed the W590S mutant had similarly low levels of [14C]cholesterol associated with apoA-I in the medium (Fig. 4D, graph). Login to comment 174 ABCA1 p.Trp590Ser As the W590S mutant had a similar impairment in its ability to transfer phosphatidylcholine to apoA-I (data not shown), the mutant does not provide a tool for dissociating the mechanisms of efflux of these two lipids. Login to comment 175 ABCA1 p.Trp590Ser ApoA-I associates with the ABCA1 W590S mutant in a temperature-dependent manner and is released from the mutant complex with kinetics similar to the release from wild-type ABCA1 (wtABCA1). Login to comment 176 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser ApoA-I associates with the ABCA1 W590S mutant in a temperature-dependent manner and is released from the mutant complex with kinetics similar to the release from wild-type ABCA1 (wtABCA1). Login to comment 177 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser A: ABCA1 and the W590S mutant display a similar temperature sensitivity of complex formation. Login to comment 178 ABCA1 p.Trp590Ser Wild-type ABCA1, the W590S mutant, or empty vector (mock)-expressing cells were incubated with 125I-apoA-I at either 37ЊC or 4ЊC for 1 h. Login to comment 181 ABCA1 p.Trp590Ser B: ApoA-I complexes between wild-type ABCA1 and the W590S mutant turn over at a similar rate. Login to comment 182 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser B: ApoA-I complexes between wild-type ABCA1 and the W590S mutant turn over at a similar rate. Login to comment 183 ABCA1 p.Trp590Ser Cells expressing ABCA1 or the W590S mutant were incubated with 125I-apoA-I at 37ЊC for 1 h and then washed with warm PBS. Login to comment 185 ABCA1 p.Trp590Ser C: ABCA1 and the W590S mutant cells release cell-associated apoA-I with similar kinetics. Login to comment 186 ABCA1 p.Trp590Ser C: ABCA1 and the W590S mutant cells release cell-associated apoA-I with similar kinetics. Login to comment 187 ABCA1 p.Trp590Ser Graphed is the amount of 125I-apoA-I released from wild-type ABCA1 and W590S-expressing cells relative to the mock cells (duplicate samples, SD) D: Only apoA-I released from wild-type ABCA1 is associated with cholesterol. Login to comment 188 ABCA1 p.Trp590Ser Graphed is the amount of 125I-apoA-I released from wild-type ABCA1 and W590S-expressing cells relative to the mock cells (duplicate samples, ϮSD) D: Only apoA-I released from wild-type ABCA1 is associated with cholesterol. Login to comment 222 ABCA1 p.Trp590Ser In our studies, we compared the dissociation behavior of wild-type ABCA1/apoA-I complexes with those formed with the W590S ABCA1 mutant. Login to comment 223 ABCA1 p.Trp590Ser In our studies, we compared the dissociation behavior of wild-type ABCA1/apoA-I complexes with those formed with the W590S ABCA1 mutant. Login to comment 224 ABCA1 p.Trp590Ser The W590S mutant, like wild-type ABCA1, was not able to form a cross-linkable complex with apoA-I at 4C. Login to comment 225 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser The W590S mutant, like wild-type ABCA1, was not able to form a cross-linkable complex with apoA-I at 4ЊC. Login to comment 226 ABCA1 p.Trp590Ser At 37ЊC, apoA-I dissociated from the W590S mutant at a rate similar to that measured using the wild-type ABCA1 (half-life of 30 versus 25 min, respectively). Login to comment 227 ABCA1 p.Trp590Ser These results suggest that the W590S mutant binds and releases apoA-I in a normal manner but that these processes are uncoupled from the transfer of cholesterol to the released apoA-I. Login to comment 228 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser These results suggest that the W590S mutant binds and releases apoA-I in a normal manner but that these processes are uncoupled from the transfer of cholesterol to the released apoA-I. Login to comment 229 ABCA1 p.Trp590Ser Our results indicate that the W590S mutant has a similar impairment in the transfer of phospholipid, and as reported by Rigot et al. (9), the mutant does not stimulate the translocation of phosphatidylserine to the outer leaflet of the plasma membrane. Login to comment 230 ABCA1 p.Trp590Ser If this mechanism were operative, it seems unlikely that the apoA-I would dissociate from the W590S mutant with the kinetics we have measured. Login to comment 231 ABCA1 p.Trp590Ser ABCA1 p.Trp590Ser If this mechanism were operative, it seems unlikely that the apoA-I would dissociate from the W590S mutant with the kinetics we have measured. Login to comment 232 ABCA1 p.Trp590Ser The behavior of the W590S mutant/apoA-I complex indicates that the cellular release of apoA-I and the transfer of cholesterol and phospholipid are separable events and thus likely distinct steps in the efflux mechanism. Login to comment 241 ABCA1 p.Trp590Ser The W590S ABCA1 mutant demonstrates that this release is not dependent on cholesterol transfer to the acceptor apolipoprotein, indicating that release and cholesterol transfer are separable events in the efflux process. Login to comment 242 ABCA1 p.Trp590Ser The W590S ABCA1 mutant demonstrates that this release is not dependent on cholesterol transfer to the acceptor apolipoprotein, indicating that release and cholesterol transfer are separable events in the efflux process. Login to comment