Recombinant expression of the MAL proteolipid, a component of glycolipid-enriched membrane microdomains, induces the formation of vesicular structures in insect cells
Recombinant expression of the MAL proteolipid, a component of glycolipid-enriched membrane microdomains, induces the formation of vesicular structures in insect cells
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DOI：
10.1074/jbc.272.29.18311
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发表时间：
1997-07-18
影响因子：
4.8
通讯作者：
Alonso, MA
中科院分区：
文献类型：
--
作者：
Puertollano, R;Li, SW;Alonso, MA
MAL 蛋白脂质已被鉴定为富含糖脂的膜微结构域的成分，该膜微结构域对上皮 Madin-Darby 犬细胞、T 淋巴细胞和髓磷脂形成细胞中的洗涤剂溶解具有抵抗力。为了研究 MAL 蛋白脂质的功能，我们在哺乳动物和昆虫细胞背景中异位表达了标记形式的 MAL，瞬时转染的 COS-7 细胞中的免疫荧光分析表明存在大囊泡结构中存在 MAL，生化分析在对 Triton X-100 溶解有抗性的膜部分中鉴定出 MAL，电子显微镜分析表明，Sf21 细胞中 MAL 的表达在形态上导致了直径从 200 至大于 700 nm 的大囊泡在细胞内的积累，而在未感染或对照感染的培养物中不存在这种情况，因此，在该异源表达系统中异位表达了 MAL足以驱动大小与哺乳动物细胞中检测到的囊泡相似的囊泡的形成，这些囊泡明显不同于由caveolin表达诱导的caveolae样囊泡，正如使用重组caveolin杆状病毒的共感染实验所证明的那样。总而言之，这些结果表明 MAL 蛋白脂质可能作为富含糖脂的膜的囊泡形成机制的一个组成部分发挥作用。
The MAL proteolipid has been identified as a component of glycolipid-enriched membrane microdomains resistant to detergent solubilization in epithelial Madin-Darby canine cells, as well as in T lymphocytes and in myelin-forming cells, To study the function of the MAL proteolipid we have ectopically expressed a tagged form of MAL in both mammalian and insect cellular backgrounds, Immunofluorescence analysis in transiently transfected COS-7 cells showed the presence of MAL in large vesicular structures, and biochemical analysis identified MAL in the fraction of membranes resistant to Triton X-100 solubilization, Electron microscopic analysis showed that the expression of MAL in Sf21 cells morphologically resulted in the intracellular accumulation of large vesicles with a diameter from 200 to greater than 700 nm that were absent in uninfected or control infected cultures, Thus, ectopic expression of MAL in this heterologous expression system was sufficient to drive the formation of vesicles with a size similar to that of the vesicles detected in mammalian cells, These vesicles were clearly different from the caveolae-like vesicles induced by caveolin expression, as evidenced by co-infection experiments using a recombinant caveolin baculovirus. Taken together, these results suggest that the MAL proteolipid might play a role as a component of the machinery of vesiculation of glycolipid-enriched membranes.