Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface
Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface
复制标题
DOI：
10.1172/jci200216000
复制
发表时间：
2002-09-01
影响因子：
15.9
通讯作者：
Hobbs, HH
中科院分区：
文献类型：
--
作者：
Graf, GA;Li, WP;Hobbs, HH
atp结合盒（ABC） G5或ABCG8的突变可引起谷固醇血症，这是一种常染色体隐性胆固醇运输疾病。为了确定ABCG5和ABCG8的作用位点，我们在培养细胞中表达重组、表位标记的小鼠ABCG5和ABCG8。ABCG5和ABCG8均发生了n链糖基化。当这两种蛋白在细胞中单独表达时，n -连接糖对内糖苷酶H （Endo H）仍然敏感。当ABCG5和ABCG8共表达时，附着的糖具有Endo h抗性和神经氨酸酶敏感性，表明蛋白质被转运到反式高尔基复合物上。成熟的、糖基化的ABCG5和ABCG8共免疫沉淀，与这两种蛋白的异源二聚化一致。Endo h敏感形式的ABCG5和ABCG8局限于内质网（ER），而成熟形式存在于培养的肝细胞的非内质网部分。免疫电镜显示，这些细胞的质膜上存在ABCG5和ABCG8。在极化的wi - b细胞中，重组ABCG5与ABCG8共表达时定位于根尖（管状）膜，而单独表达时不定位。据我们所知，这是第一次直接证明ABC半转运体运输到细胞表面需要其二聚化伙伴的存在。
Mutations in either ATP-binding cassette (ABC) G5 or ABCG8 cause sitosterolemia, an autosomal recessive disorder of sterol trafficking. To determine the site of action of ABCG5 and ABCG8, we expressed recombinant, epitope-tagged mouse ABCG5 and ABCG8 in cultured cells. Both ABCG5 and ABCG8 underwent N-linked glycosylation. When either protein was expressed individually in cells, the N-linked sugars remained sensitive to Endoglycosidase H (Endo H). When ABCG5 and ABCG8 were coexpressed, the attached sugars were Endo H-resistant and neuraminidase-sensitive, indicating that the proteins were transported to the trans-Golgi complex. The mature, glycosylated forms of ABCG5 and ABCG8 coimmunoprecipitated, consistent with heterodimerization of these two proteins. The Endo H-sensitive forms of ABCG5 and ABCG8 were confined to the endoplasmic reticulum (ER), whereas the mature forms were present in non-ER fractions in cultured hepatocytes. immunoelectron microscopy revealed ABCG5 and ABCG8 on the plasma membrane of these cells. In polarized WIF-B cells, recombinant ABCG5 localized to the apical (canalicular) membrane when coexpressed with ABCG8, but not when expressed alone. To our knowledge this is the first direct demonstration that trafficking of an ABC half-transporter to the cell surface requires the presence of its dimerization partner.