A PEST deletion mutant of ABCA1 shows impaired internalization and defective cholesterol efflux from late endosomes
A PEST deletion mutant of ABCA1 shows impaired internalization and defective cholesterol efflux from late endosomes
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DOI：
10.1074/jbc.m505566200
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发表时间：
2005-08-12
影响因子：
4.8
通讯作者：
Tall, AR
中科院分区：
文献类型：
--
作者：
Chen, WG;Wang, N;Tall, AR
ATP结合盒转运体A1（ABCA 1）促进细胞胆固醇和磷脂流出至apoA-I。我们之前描述了ABCA 1中的细胞质PEST序列，并表明PEST序列的缺失导致ABCA 1细胞表面浓度显着增加。在目前的研究中，我们评估了PEST序列缺失的ABCA 1可能显示有缺陷的内化和运输到晚期内体/溶酶体的假设。通过莫能菌素处理和细胞表面生物锡基化评估，与野生型ABCA 1（ABCA 1-wt）相比，PEST序列缺失的ABCA 1（ABCA 1-dPEST）的内化率显著降低。ABCA 1-wt的免疫荧光共聚焦显微镜显示质膜定位和与LAMP 2在晚期内体中的实质性共定位。相反，ABCA 1-dPEST显示出更突出的质膜定位，但与LAMP 2的共定位很少。为了评估晚期内体的胆固醇流出，HEK 293细胞与清道夫受体A（SR-A）瞬时共转染，并与[H-3]胆固醇/乙酰基低密度脂蛋白（acLDL）孵育。尽管ABCA 1-dPEST在细胞表面标记后显示出比ABCA 1-wt更高的胆固醇流出（[H-3]胆固醇/α LDL，在不存在SR-A共转染的情况下），但其在晚期内体标记后显示出受损的胆固醇流出（[3 H]胆固醇/acLDL，在存在SR-A的情况下）。因此，PEST序列的缺失导致ABCA 1的内化减少和晚期内体胆固醇池的胆固醇流出减少，这提供了ABCA 1的内化和运输在介导胆固醇从细胞内胆固醇池流出中具有重要功能的证据。
ATP-binding cassette transporter A1 (ABCA1) promotes the efflux of cellular cholesterol and phospholipids to apoA-I. We described previously a cytoplasmic PEST sequence in ABCA1 and showed that deletion of the PEST sequence results in a prominent increase in the cell surface concentration of ABCA1. In the current study we evaluated the hypothesis that the PEST sequence-deleted ABCA1 might display defective internalization and trafficking to the late endosomes/lysosomes. As assessed by monensin treatment and cell surface bio-tinylation, the internalization rate of PEST sequence-deleted ABCA1 (ABCA1-dPEST) was markedly decreased compared with wild-type ABCA1 (ABCA1-wt). Immunofluorescence confocal microscopy of ABCA1-wt showed both plasma membrane localization and substantial co-localization with LAMP2 in late endosomes. In contrast, ABCA1-dPEST showed more prominent plasma membrane localization but little co-localization with LAMP2. To assess cholesterol efflux from late endosomes, HEK293 cells were transiently co-transfected with scavenger receptor A (SR-A) and incubated with [H-3] cholesterol/acetyl low density lipoprotein (acLDL). Although ABCA1-dPEST showed higher cholesterol efflux than did ABCA1-wt following cell surface labeling ([H-3] cholesterol/alpha LDL in the absence of SR-A co-transfection), it showed impaired cholesterol efflux after late endosomal labeling ([3H] cholesterol/acLDL in the presence of SR-A). Thus, deletion of the PEST sequence leads to a decrease in the internalization of ABCA1 and decreased cholesterol efflux from late endosomal cholesterol pools, providing evidence that the internalization and trafficking of ABCA1 is functionally important in mediating cholesterol efflux from intracellular cholesterol pools.