Millán J, Puertollano R, Fan L, Alonso M A
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, Cantoblanco, Madrid, Spain.
Biochem Biophys Res Commun. 1997 Apr 28;233(3):707-12. doi: 10.1006/bbrc.1997.6530.
The MAL proteolipid and caveolin have been identified as components of internal detergent-insoluble membrane microdomains enriched in glycolipids and cholesterol. We have addressed the study of the glycolipid-enriched membranes in cells expressing endogenously only either MAL (Jurkat T cells) or caveolin (epithelial A498 cells) and in polarized MDCK cells which express both proteins simultaneously. Subcellular fractionation by centrifugation to equilibrium in sucrose gradients of Triton X-100 cell extracts from Jurkat and A498 cells revealed that MAL and caveolin are incorporated in detergent-insoluble buoyant membranes independently of the expression of each other and indicated the existence in these cells of insoluble membrane microdomains with either MAL or caveolin. Immunofluorescence analysis in MDCK cells indicated that both MAL and caveolin were located in the Golgi region, whereas caveolin was found in addition at the cell surface. Biochemical analysis in these cells revealed the existence of distinct membrane microenvironments differentially susceptible to detergent solubilization containing either internal MAL or internal plus surface caveolin. The observed heterogeneity within the internal glycolipid-enriched membrane fraction suggests the existence of distinct specialized lipid microenvironments in MDCK cells.
MAL蛋白脂质和小窝蛋白已被鉴定为富含糖脂和胆固醇的内部去污剂不溶性膜微区的组成成分。我们研究了仅内源性表达MAL（Jurkat T细胞）或小窝蛋白（上皮A498细胞）的细胞以及同时表达这两种蛋白的极化MDCK细胞中富含糖脂的膜。通过在Jurkat和A498细胞的Triton X-100细胞提取物的蔗糖梯度中进行平衡离心进行亚细胞分级分离，结果显示MAL和小窝蛋白独立于彼此的表达而被整合到去污剂不溶性浮力膜中，这表明在这些细胞中存在含有MAL或小窝蛋白的不溶性膜微区。MDCK细胞中的免疫荧光分析表明，MAL和小窝蛋白均位于高尔基体区域，而小窝蛋白还存在于细胞表面。对这些细胞的生化分析揭示了存在不同的膜微环境，这些微环境对去污剂溶解的敏感性不同，其中含有内部的MAL或内部加表面的小窝蛋白。在富含内部糖脂的膜部分中观察到的异质性表明MDCK细胞中存在不同的特殊脂质微环境。
