Subcellular localization and detergent solubility of MVP17/rMAL, a lipid raft-associated protein in oligodendrocytes and myelin.
Subcellular localization and detergent solubility of MVP17/rMAL, a lipid raft-associated protein in oligodendrocytes and myelin.
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MVP17/rMAL（少突胶质细胞和髓磷脂中的脂筏相关蛋白）的亚细胞定位和洗涤剂溶解度。
DOI：
10.1002/jnr.10283
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Pfeiffer,StevenE
中科院分区：
文献类型：
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作者：
Kim,Taeyoon;Pfeiffer,StevenE
不溶于洗涤剂、富含鞘糖脂胆固醇的微区（脂筏）与蛋白质运输和信号转导有关。之前，我们在少突胶质细胞和髓鞘中鉴定了脂筏相关的、整合的膜蛋白髓鞘囊泡蛋白17 kDa（MVP 17）/rMAL。在这里，我们检查了天然和重组MVP 17/rMAL在转染的少突胶质细胞和COS-7细胞以及纯化的髓鞘中的亚细胞定位和/或去污剂不溶性。与我们之前关于MVP 17/rMAL在两性离子去污剂3-[（3-氯酰胺丙基）-二甲基铵基]-1-丙烷-磺酸盐（CHAPS）中的不溶性的报道一致，来自培养物中纯化的髓鞘和少突胶质细胞的MVP 17/rMAL在4°C下用非离子去污剂Triton X-100提取时大部分不溶，并且在蔗糖梯度超离心中漂浮至低密度，但在37°C下变为去污剂可溶的。通过免疫荧光显微镜获得的转染到少突胶质细胞和COS-7细胞中的表位标记的MVP 17/rMAL表达的数据与该蛋白的N-和C-末端均面向细胞质的模型一致。突变分析鉴定了MVP 17/rMAL的结构域，其对亚细胞定位和其去污剂溶解性谱是重要的。特别是，环II的插入突变阻止了突变蛋白插入COS-7细胞的质膜，并使其不溶于TX-100。在COS-7细胞中表达MVP 17/rMAL全长构建体导致转染的COS-7细胞增大，这与rMAL在囊泡运输中的作用一致。© 2002 Wiley利斯公司
Detergent‐insoluble, glycosphingolipid‐cholesterol‐enriched microdomains (lipid rafts) have been implicated in both protein trafficking and signal transduction. Previously we identified in oligodendrocytes and myelin the lipid raft‐associated, integral membrane protein myelin vesicular protein of 17 kDa (MVP17)/rMAL. Here we have examined the subcellular localization and/or detergent insolubility of native and recombinant MVP17/rMAL in transfected oligodendrocytes and COS‐7 cells and purified myelin. Consistent with our previous report regarding the insolubility of MVP17/rMAL in the zwitterionic detergent 3‐[(3‐chloramidopropyl)‐dimethylammonio]‐1‐propane‐sulfonate (CHAPS), MVP17/rMAL from purified myelin and oligodendrocytes in culture was mostly insoluble upon extraction at 4°C with the non‐ionic detergent Triton X‐100 and floated to a low density in sucrose gradient ultracentrifugation, but became detergent soluble at 37°C. Data obtained by immunofluorescence microscopy of the expression of epitope‐tagged MVP17/rMAL transfected into oligodendrocytes and COS‐7 cells were consistent with a model in which both the N‐ and C‐termini of this protein face the cytoplasm. Mutational analysis identified domains of MVP17/rMAL important for its subcellular localization and for its detergent solubility profile. In particular, insertional mutagenesis of loop II prevented the insertion of the mutant protein into the plasma membrane of COS‐7 cells and rendered it insoluble in TX‐100. Expression of full‐length constructs of MVP17/rMAL in COS‐7 cells resulted in an enlargement of transfected COS‐7 cells, consistent with a proposed role of rMAL in vesicular trafficking. © 2002 Wiley‐Liss, Inc.