Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona, United States of America.
PLoS One. 2011 Mar 31;6(3):e18150. doi: 10.1371/journal.pone.0018150.
One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.
METHODOLOGY/PRINCIPAL FINDINGS: Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta'), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.
alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases.
埃及伊蚊中肠上皮细胞的早期事件之一是粗面内质网（RER）涡旋结构的动态重排，与血液消化的开始同时发生。基于我们之前的研究表明，在进食氨基酸餐后，埃及伊蚊中的 TOR 信号被诱导，我们使用蛋白质组学和 RNAi 来鉴定有助于 RER 涡旋形成的中肠上皮细胞蛋白。
方法/主要发现：成年雌性埃及伊蚊被单独维持在糖上（未进食），或喂食氨基酸餐，然后通过电子显微镜和蛋白质生物化学分析中肠上皮细胞。中肠上皮细胞中 RER 涡旋的大小和数量在进食后明显减少，并且几种含有 KDEL 的蛋白质的表达水平发生改变。LC-MS/MS 质谱法用于分析未进食和氨基酸喂养的蚊子中分离的中肠微粒体蛋白，在鉴定的 127 种蛋白质中，选择了 8 种作为候选涡旋形成蛋白。3 种候选蛋白是 COPI 衣壳蛋白亚基（alpha、beta、beta'），它们似乎都在未进食蚊子的微粒体部分中存在更高的水平。使用 RNAi 敲低 alpha-COPI 的表达，电子显微镜显示，在未进食的蚊子中，RER 涡旋的大小和数量都显著减少，而且在进食的蚊子中，扩张的 RER 区域很常见。最后，虽然 alpha-COPI 的缺乏对 3 小时血餐后（PBM）早期胰蛋白酶蛋白的合成或分泌没有影响，但在 24 小时 PBM 时晚期蛋白酶的表达完全被阻断。
alpha-COPI 被发现是未进食的埃及伊蚊中肠上皮细胞中 RER 涡旋形成所必需的，也是晚期中肠蛋白酶表达所必需的。
