ABCMdb

PMID: 16984907

Landry YD, Denis M, Nandi S, Bell S, Vaughan AM, Zha X
ATP-binding cassette transporter A1 expression disrupts raft membrane microdomains through its ATPase-related functions.
J Biol Chem. 2006 Nov 24;281(47):36091-101. Epub 2006 Sep 19., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCA1 p.Ala937Val Cholesterol content in the plasma membraneis,however,notaltered.Moreover,weprovideevidence that a non-functional ABCA1 with mutation in an ATP-binding domain,A937V,failstoredistributecholesterol,sphingomyelin,or caveolin. Login to comment 4 ABCA1 p.Ala937Val A937V also fails to influence Akt activation. Login to comment 110 ABCA1 p.Ala937Val Cy3-Transferrin Labeling of EYFP-Caveolin-1-transfected Cells-Mock, ABCA1, and A937V mutant cells were grown in 35-mm glass-bottom dishes and transfected with EYFP-caveolin-1 as described above. Login to comment 118 ABCA1 p.Ala937Val Mock, ABCA1, and A937V mutant cells were grown to 80% confluence in 20-cm plates (five per cell line), and incubated in DMEM with 1 mg/ml BSA and 10 nM mifepristone for 18 h to induce ABCA1 expression. Login to comment 132 ABCA1 p.Ala937Val Subcellular [3 H]Cholesterol Distribution-Mock, ABCA1, and A937V mutant cells were grown to 80% confluence in 20-cm plates (two for each cell line). Login to comment 145 ABCA1 p.Ala937Val Briefly, mock, ABCA1, and A937V mutant cells were grown to 80% confluence in 20-cm plates. Login to comment 168 ABCA1 p.Ala937Val A, ABCA1 expression in Mock, ABCA1 and ABCA1 mutant, A937V, before (lane 1-3) and after over night incubation with 10 nM mifepristone (lane 4-12). Login to comment 191 ABCA1 p.Ala937Val ABCA1-expressing cells, however, seemed to have slightly less cholesterol in the endoplasmic reticulum (fractions 9 and 10) than Mock or A937V cells, which may reflect higher cholesterol mobility from internal membranes to the plasma membrane upon ABCA1 expression. Login to comment 192 ABCA1 p.Ala937Val ABCA1-expressing cells, however, seemed to have slightly less cholesterol in the endoplasmic reticulum (fractions 9 and 10) than Mock or A937V cells, which may reflect higher cholesterol mobility from internal membranes to the plasma membrane upon ABCA1 expression. Login to comment 193 ABCA1 p.Ala937Val We also included a BHK cell line that expresses a mutant form of ABCA1 (A937V) upon mifepristone induction. Login to comment 194 ABCA1 p.Ala937Val We also included a BHK cell line that expresses a mutant form of ABCA1 (A937V) upon mifepristone induction. Login to comment 195 ABCA1 p.Ala937Val Cholesterol distribution in A937V-expressing cells was also identical to that of wt ABCA1-expressing or Mock cells (Fig. 2, FIGURE 2. Login to comment 196 ABCA1 p.Ala937Val Cholesterol distribution in A937V-expressing cells was also identical to that of wt ABCA1-expressing or Mock cells (Fig. 2, FIGURE 2. Login to comment 197 ABCA1 p.Ala937Val A, ABCA1 distribution in Mock, ABCA1, and A937V cells. Login to comment 198 ABCA1 p.Ala937Val A, ABCA1 distribution in Mock, ABCA1, and A937V cells. Login to comment 201 ABCA1 p.Ala937Val B, Mock, ABCA1, and A937V cells were stained with filipin, and fluorescent images were taken under identical conditions. Login to comment 202 ABCA1 p.Ala937Val B, Mock, ABCA1, and A937V cells were stained with filipin, and fluorescent images were taken under identical conditions. Login to comment 207 ABCA1 p.Ala937Val A937V contains a naturally occurring mutation in its first ATP-binding domain and is not able to mediate cholesterol efflux (Fig. 1B). Login to comment 208 ABCA1 p.Ala937Val A937V contains a naturally occurring mutation in its first ATP-binding domain and is not able to mediate cholesterol efflux (Fig. 1B). Login to comment 210 ABCA1 p.Ala937Val Mock, ABCA1, and A937V cells were treated with ice-cold Triton X-100 (1%) for 30 min. Login to comment 211 ABCA1 p.Ala937Val Mock, ABCA1, and A937V cells were treated with ice-cold Triton X-100 (1%) for 30 min. Login to comment 220 ABCA1 p.Ala937Val To address this, we used BHK cells expressing A937V. Login to comment 221 ABCA1 p.Ala937Val ABCA1 p.Ala937Val The cold Triton X-100-extractable cholesterol fraction in A937V-expressing cells was identical to that in Mock cells (Fig. 3, A and B), indicating that cholesterol redistribution from raft to non-rafts requires functional ABCA1 protein. Login to comment 222 ABCA1 p.Ala937Val The cold Triton X-100-extractable cholesterol fraction in A937V-expressing cells was identical to that in Mock cells (Fig. 3, A and B), indicating that cholesterol redistribution from raft to non-rafts requires functional ABCA1 protein. Login to comment 226 ABCA1 p.Ala937Val Once again, the A937V mutant failed to redistribute [3 H]sphingomyelin, consistent with its incapability to redistribute cholesterol in these cells. Login to comment 227 ABCA1 p.Ala937Val Once again, the A937V mutant failed to redistribute [3 H]sphingomyelin, consistent with its incapability to redistribute cholesterol in these cells. Login to comment 238 ABCA1 p.Ala937Val We transiently transfected Mock, ABCA1, and A937V cells with YFP-caveolin. Login to comment 239 ABCA1 p.Ala937Val We transiently transfected Mock, ABCA1, and A937V cells with YFP-caveolin. Login to comment 242 ABCA1 p.Ala937Val ABCA1 p.Ala937Val BHK cells induced to express either empty vector (Mock), ABCA1 (ABCA1), or ABCA1 mutant A937V (A937V) cells were incubated with mifepristone overnight and subjected to 1% cold Triton X-100 extraction for 30 min on ice. Login to comment 243 ABCA1 p.Ala937Val ABCA1 p.Ala937Val BHK cells induced to express either empty vector (Mock), ABCA1 (ABCA1), or ABCA1 mutant A937V (A937V) cells were incubated with mifepristone overnight and subjected to 1% cold Triton X-100 extraction for 30 min on ice. Login to comment 248 ABCA1 p.Ala937Val C and D, Mock, ABCA1, and A937V cells were radiolabeled with 1 ␮Ci/ml [3 H]choline for 24 h, chilled on ice for 30 min prior to 1% Triton X-100 extraction at 4 °C. Login to comment 249 ABCA1 p.Ala937Val C and D, Mock, ABCA1, and A937V cells were radiolabeled with 1 òe;Ci/ml [3 H]choline for 24 h, chilled on ice for 30 min prior to 1% Triton X-100 extraction at 4 &#b0;C. Login to comment 277 ABCA1 p.Ala937Val ABCA1 p.Ala937Val Furthermore, when we tested ABCA1 mutant A937V, we found that A937V was mainly localized on the plasma membrane and expressed at levels similar to wild-type ABCA1 (Fig. 6C). Login to comment 279 ABCA1 p.Ala937Val YFP-caveolin in A937V cells was mostly clustered, identical to that in Mock cells. Login to comment 280 ABCA1 p.Ala937Val This is consistent with our observations presented above that A937V is defective in redistributing rafts (Fig. 3). Login to comment 291 ABCA1 p.Ala937Val ABCA1 p.Ala937Val We therefore performed experiments using a non- detergent-based method, Optiprep gradient floatation, on Mock, ABCA1, and A937V cells (Fig. 7A). Login to comment 293 ABCA1 p.Ala937Val A minor portion of caveolin (ϳ5%) was found in fractions 7-9, co-localizing with clathrin, a non-raft maker, in Mock and A937V cells. Login to comment 294 ABCA1 p.Ala937Val However, there was much more caveolin in the non-raft fractions (20%) in ABCA1 cells than either Mock or A937V cells (Fig. 7B). Login to comment 295 ABCA1 p.Ala937Val There was no significant difference between Mock and A937V cells. Login to comment 300 ABCA1 p.Ala937Val BHK cells expressing either wt ABCA1 or A937V were transfected with YFP-caveolin and immunostained for ABCA1. Login to comment 305 ABCA1 p.Ala937Val Mock, ABCA1, and A937V cells were induced overnight with mifepristone, and membrane fractions were isolated as described under "Materials and Methods." Login to comment 307 ABCA1 p.Ala937Val B, the Western blots were scanned and analyzed for Mock, ABCA1, and A937V cells. Login to comment 313 ABCA1 p.Ala937Val Expression of A937V once again does not alter Akt activation (Fig. 8A). Login to comment 330 ABCA1 p.Ala937Val Mock, ABCA1, and A937V cells were induced and serum-starved overnight before EGF treatment. Login to comment 358 ABCA1 p.Ala937Val Most importantly, we found that a functional nucleotide-binding domain in ABCA1 is required for non-raft expansion, because the A937V mutant failed to induce any cholesterol, sphingomyelin, or caveolin redistribution. Login to comment 359 ABCA1 p.Ala937Val A937V mutant also failed to impair Akt phosphorylation. Login to comment 377 ABCA1 p.Ala937Val Our observations with the A937V mutant seem to support this later notion. Login to comment 378 ABCA1 p.Ala937Val Our observations with the A937V mutant seem to support this later notion. Login to comment