Baculovirus-based expression of mammalian caveolin in Sf21 insect cells A model system for the biochemical and morphological study of caveolae biogenesis
Baculovirus-based expression of mammalian caveolin in Sf21 insect cells A model system for the biochemical and morphological study of caveolae biogenesis
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DOI：
10.1074/jbc.271.45.28647
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发表时间：
1996-11-08
影响因子：
4.8
通讯作者：
Lisanti, MP
中科院分区：
文献类型：
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作者：
Li, SW;Song, KS;Lisanti, MP
小泡最初在形态学上被定义为位于或靠近质膜的50-100 nm无包被的囊状细胞器。小泡蛋白是一种21 kDa的囊泡整体膜蛋白，是体内小泡膜的主要蛋白质成分。小泡蛋白与自身相互作用形成高分子质量的低聚物，表明它可能在小泡膜的形成中起结构作用。然而，在体内，小窝蛋白的重组表达是否足以产生小窝膜仍存在争议。为了直接解决这个问题，我们采用了一种不同的实验方法，利用异源表达系统。在这里，我们利用基于杆状病毒的载体在Sf21昆虫细胞中重组表达了哺乳动物小窝蛋白。小窝蛋白的两种同工异构体已被确定，它们的极端N端不同；α -洞穴蛋白含有残基1-178，β -洞穴蛋白含有残基32-178。在Sf21昆虫细胞中重组表达后，α -和β -小窝蛋白均形成了与天然小窝蛋白大小相同的抗sds高分子量低聚物。形态学上，两种小泡蛋白异构体的表达均导致细胞内聚集了直径在50 - 120 nm （80.3 +/- 14.8 nm）之间的均匀小泡大小的囊泡。这表明每个小窝蛋白异构体都可以独立地产生这些结构，并且小窝蛋白残基1-31不需要这个过程。利用小泡蛋白作为标记蛋白，采用无洗涤剂的方法从哺乳动物细胞中纯化小泡，我们从昆虫细胞中纯化了这些重组小泡蛋白诱导的小泡。这些纯化的重组囊泡：(1)具有与哺乳动物囊泡相同的浮力密度；（ii）全片电镜显示类似于50-100 nm的膜状结构；（iii）通过Western blotting检测，含有接近95%的重组表达的caveolin蛋白。用抗小窝蛋白IgG对这些结构进行免疫标记，证实它们含有小窝蛋白。因此，在这个异源系统中，小泡蛋白的异位过表达足以驱动小泡样囊泡的形成。进一步的功能分析表明，当与两种重组杆状病毒共感染在昆虫细胞中共表达时，小窝蛋白能够与已知的小窝蛋白相互作用蛋白Ha-Ras相互作用。总之，我们的研究结果表明，基于杆状病毒在昆虫细胞中表达小窝蛋白为研究小窝的生物发生提供了一个有吸引力的实验系统。
Caveolae were originally defined morphologically as 50-100 nm noncoated vesicular organelles located at or near the plasma membrane. Caveolin, a vesicular integral membrane protein of 21 kDa, is a principal protein component of caveolae membranes in vivo. Caveolin interacts with itself to form high molecular mass oligomers, suggesting that it might play a structural role in the formation of caveolae membranes. However, it remains controversial whether recombinant expression of caveolin is necessary or sufficient to generate caveolae membranes in vivo. To directly address this issue, we have taken a different experimental approach by exploiting a heterologous expression system. Here, we have recombinantly expressed mammalian caveolin in Sf21 insect cells using baculovirus-based vectors. Two isoforms of caveolin have been identified that differ at their extreme N terminus; alpha-caveolin contains residues 1-178, and beta-caveolin contains residues 32-178. After recombinant expression in Sf21 insect cells, both alpha- and beta-caveolin formed SDS-resistant high molecular mass oligomers of the same size as native caveolin. Morphologically, expression of either caveolin isoform resulted in the intracellular accumulation of a homogeneous population of caveolae-sized vesicles with a diameter between 50 and 120 nm (80.3 +/- 14.8 nm). This indicates that each caveolin isoform can independently generate these structures and that caveolin residues 1-31 are not required for this process. Using caveolin as a marker protein and a detergent-free procedure to purify caveolae from mammalian cells, we purified these recombinant caveolin-induced vesicles from insect cells. These purified recombinant vesicles: (i) have the same buoyant density as mammalian caveolae; (ii) appear as similar to 50-100 nm membranous structures by whole mount electron microscopy; and (iii) contain similar to 95% of the recombinantly expressed caveolin protein by Western blotting. Immuno-labeling of these structures with anti-caveolin IgG confirmed that they contain caveolin. Thus, ectopic overexpression of caveolin in this heterologous system is sufficient to drive th formation of caveolae-like vesicles. Further functional analysis demonstrated that caveolin was capable of interacting with a known caveolin-interacting protein, Ha-Ras, when coexpressed in insect cells by co-infection with two recombinant baculoviruses. Taken together, our results demonstrate that baculovirus-based expression of caveolin in insect cells provides an attractive experimental system for studying the biogenesis of caveolae.