ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane
ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane
复制标题
DOI：
10.1091/mbc.11.11.3675
复制
发表时间：
2000-11-01
影响因子：
3.3
通讯作者：
Rudel, LL
中科院分区：
文献类型：
--
作者：
Joyce, CW;Shelness, GS;Rudel, LL
第二种形式的酶酰基辅酶A：胆固醇酰基转移酶，ACAT2，已被确定。为了探索两种ACAT酶具有独立功能的假设，我们检测了ACAT1和ACAT2的膜拓扑结构。一个糖基化报告基因和FLAG表位标签序列被附加到一系列截断的ACAT cdna上。在每个预测的跨膜结构域之后。融合构建体在体外组装到微粒体膜中，并根据糖基化位点的使用和外源蛋白酶的可及性确定拓扑结构。通过渗透转染细胞的免疫荧光显微镜检测构建物中c端FLAG表位的可及性。ACAT1和ACAT2均跨越膜5次，其N端在细胞质中，C端在内质网管中。第四个跨膜结构域位于每种蛋白质的不同区域，将假定的活性位点ACAT1丝氨酸（Ser(269)）放置在细胞质中，而类似的ACAT2残基（Ser(249)）位于内质网腔中。这些丝氨酸的突变使ACAT酶失活。该结果与ACAT2形成胆固醇酯可能与脂蛋白颗粒组装和分泌耦合的假设一致，而ACAT1可能主要起维持细胞内游离胆固醇和酯化胆固醇平衡的作用。
A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated. after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser(269)) in the cytosol and the analogous residue in ACAT2 (Ser(249)) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.