Identification of two regions in apolipoprotein B100 that are exposed on the cytosolic side of the endoplasmic reticulum membrane.
Identification of two regions in apolipoprotein B100 that are exposed on the cytosolic side of the endoplasmic reticulum membrane.
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DOI：
10.1083/jcb.141.3.585
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发表时间：
1998-05-04
期刊：
影响因子：
--
通讯作者：
Dixon JL
中科院分区：
文献类型：
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作者：
Du X;Stoops JD;Mertz JR;Stanley CM;Dixon JL
载脂蛋白B100（载脂蛋白B）在毛地黄皂苷透化的HepG 2细胞的蛋白酶保护试验表明，载脂蛋白B的多个域暴露于胞质溶胶通过广泛的分泌途径的一部分。通过免疫细胞化学使用识别链球菌溶血素-O（STP-O）和皂苷透化的HepG 2细胞中apoB的特异性结构域的抗体，证实了apoB在分泌途径中的细胞内取向。在STP-O处理的细胞中，分泌途径隔室（p63，p53和半乳糖基转移酶）中标记蛋白的管腔表位未被抗体染色，但在皂苷处理的细胞中被明亮地染色，证实了在STP-O处理的细胞中内膜未穿孔。识别氨基酸3221-3240的抗apoB肽抗体（B4）在靠近核膜处引起强烈染色，而在STP-O透化细胞的整个分泌途径中不太强烈。STP-O-和皂苷处理的细胞中用该抗体染色是相似的，表明apoB中的该表位在apoB合成的位点处暴露于胞质溶胶，并且贯穿大部分剩余的分泌途径。用识别apoB中氨基酸690-797（79-91 kD）的单克隆抗体CC3.4获得了表明胞质取向的类似结果。针对人LDL制备的两种多克隆抗体和识别apoB中氨基酸1878-2148（D7.2）和3214-3506（B1 B6）的两种单克隆抗体在STP-O处理的细胞中没有产生针对apoB的强网状信号。抗低密度脂蛋白和B1 B6抗体在STP-O处理的细胞中产生几乎相同的点状图案，这些图案与溶菌酶的膜标记物LAMP-1重叠。这些观察结果表明apoB的B1 B6表位暴露在溶酶体表面。结果确定了两个特定区域的载脂蛋白B暴露于分泌途径中的胞质溶胶。
Protease protection assays of apolipoprotein B100 (apoB) in digitonin-permeabilized HepG2 cells indicated that multiple domains of apoB are exposed to the cytosol through an extensive portion of the secretory pathway. The intracellular orientation of apoB in the secretory pathway was confirmed by immunocytochemistry using antibodies recognizing specific domains of apoB in streptolysin-O (STP-O)– and saponin-permeabilized HepG2 cells. Lumenal epitopes on marker proteins in secretory pathway compartments (p63, p53, and galactosyltransferase) were not stained by antibodies in STP-O–treated cells, but were brightly stained in saponin-treated cells, confirming that internal membranes were not perforated in STP-O–treated cells. An anti-apoB peptide antibody (B4) recognizing amino acids 3221–3240 caused intense staining in close proximity to the nuclear membrane, and less intensely throughout the secretory pathway in STP-O–permeabilized cells. Staining with this antibody was similar in STP-O– and saponin-treated cells, indicating that this epitope in apoB is exposed to the cytosol at the site of apoB synthesis and throughout most of the remaining secretory pathway. Similar results indicating a cytosolic orientation were obtained with monoclonal antibody CC3.4, which recognizes amino acids 690–797 (79–91 kD) in apoB. Two polyclonal antibodies made to human LDL and two monoclonal antibodies recognizing amino acids 1878–2148 (D7.2) and 3214–3506 (B1B6) in apoB did not produce a strong reticular signal for apoB in STP-O–treated cells. The anti-LDL and B1B6 antibodies produced almost identical punctate patterns in STP-O–treated cells that overlapped with LAMP-1, a membrane marker for lysosomes. These observations suggest that the B1B6 epitope of apoB is exposed on the surface of the lysosome. The results identify two specific regions in apoB that are exposed to the cytosol in the secretory pathway.