Renninger S, Backendorf E, Kreimer G
Botanisches Institut, Universität zu Köln, Cologne, Germany.
Planta. 2001 May;213(1):51-63. doi: 10.1007/s004250000473.
Despite the well-characterized function of the green-algal eyespot apparatus as a combined absorption/reflection screen for the photoreceptor for phototaxis, little is known about the proteins involved in the formation of this complex organelle. We therefore purified the carotenoid-rich lipid globules, which are the most conspicuous component of the eyespot sensu strictu from Spermatozopsis similis Preisig et Melkonian. Electron microscopy and an average carotenoid:chlorophyll ratio of 51, confirmed the high purity of the fraction. The diameter of isolated globules (approx. 112 nm) fell within their in vivo range (90-120 nm). Absorption spectra in aqueous media peaked at 535 nm. The predominant carotenoids were beta/psi-, beta, beta- and delta-carotene. Freeze-fracture studies with cells and whole-mount electron microscopy of isolated globules demonstrated regularly arranged particles at the globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophresis revealed specific enrichment of 10 tightly bound major proteins and several minor proteins with the globules. Proteases were used to analyze their topology and function. Upon treatment with thermolysin, globules were released from a fraction enriched in isolated eyespot apparatuses. Major proteins of these globules, and those treated with thermolysin after isolation, were identical. However, the purified proteins were sensitive to thermolysin, indicating that domains of them are normally hidden in the globule matrix. In contrast, pronase degraded all globule-associated proteins in situ. These globules were not stable and easily fused, whereas thermolysin-treated globules were relatively stable. Lipase did not affect globule stability. These results indicate that the five thermolysin-resistant proteins (apparent Mr values: 56, 52, 32, 29, 27 kDa) are close to the surface and might be crucial for globule stabilization, whereas the thermolysin-accessible proteins are probably involved in globule/globule interactions and/or globule/eyespot-membrane interactions.
尽管绿藻眼点装置作为趋光性光感受器的吸收/反射组合屏的功能已得到充分表征，但对于参与形成这种复杂细胞器的蛋白质却知之甚少。因此，我们从相似精子藻（Preisig和Melkonian）中纯化了富含类胡萝卜素的脂质小球，它们是狭义眼点最显著的成分。电子显微镜和平均类胡萝卜素：叶绿素比为51，证实了该组分的高纯度。分离出的小球直径（约112纳米）在其体内范围（90 - 120纳米）内。在水性介质中的吸收光谱在535纳米处达到峰值。主要类胡萝卜素为β/ψ -、β、β - 和δ - 胡萝卜素。对细胞进行的冷冻断裂研究以及对分离出的小球进行整装电子显微镜观察表明，小球表面有规则排列的颗粒。十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示，小球特异性富集了10种紧密结合的主要蛋白质和几种次要蛋白质。使用蛋白酶分析它们的拓扑结构和功能。用嗜热菌蛋白酶处理后，小球从富含分离出的眼点装置的组分中释放出来。这些小球的主要蛋白质，以及分离后用嗜热菌蛋白酶处理的小球的主要蛋白质，是相同的。然而，纯化后的蛋白质对嗜热菌蛋白酶敏感，表明它们的结构域通常隐藏在小球基质中。相比之下，链霉蛋白酶在原位降解了所有与小球相关的蛋白质。这些小球不稳定且容易融合，而经嗜热菌蛋白酶处理的小球相对稳定。脂肪酶不影响小球稳定性。这些结果表明，五种抗嗜热菌蛋白酶的蛋白质（表观分子量值：56、52、32、29、27 kDa）靠近表面，可能对小球稳定至关重要，而可被嗜热菌蛋白酶作用的蛋白质可能参与小球/小球相互作用和/或小球/眼点膜相互作用。
