Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli.
Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli.
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大肠杆菌的有效体外检测系统揭示了质子动力依赖性和非依赖性蛋白质易位。
DOI：
10.1016/s0021-9258(18)94246-8
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
S. Mizushima
中科院分区：
文献类型：
--
作者：
H. Yamada;H. Tokuda;S. Mizushima
将大肠杆菌原生质球制备的倒膜囊泡通过蔗糖梯度离心分离，获得分泌蛋白高效定量易位的囊泡制剂。 OmpA 和不可切割模型蛋白不可切割 OmpF-Lpp 的易位几乎在 2-3 分钟内完成，而 OmpF-Lpp（一种嵌合分泌蛋白）的易位需要 20 分钟才能完成。然后用这三种蛋白质检查体外易位的质子动力(Δμ～H+)的要求。添加羰基氰化物间氯苯腙(CCCP)或使用缺乏F 1-ATP酶的剥离膜囊泡时，所有这些蛋白质的转位均被显着抑制，这表明Δμ~H+通常参与转位反应。 OmpF-Lpp 的抑制作用是完全的，而大量不可切割的 OmpF-Lpp 和 OmpA 在高浓度的羰基氰化物间氯苯腙存在下，即使在剥离膜囊泡的情况下也会以较慢的速率易位。 Δμ~H+非依赖性易位被不可水解的ATP类似物抑制。这些结果表明，尽管OmpF-Lpp的易位必须需要Δμ～H+，但后两种蛋白质不仅可以以Δμ～H+依赖性方式易位，而且可以以不依赖Δμ～H+的方式易位。
Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained. The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2–3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion. The requirement of the proton motive force (Δ μ˜ H+) for in vitro translocation was then examined with these three proteins. The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F 1-ATPase were used, suggesting that Δ μ˜ H+ generally participates in the translocation reaction. The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone. The Δ μ˜ H+-independent translocation was inhibited by a nonhydrolyzable ATP analogue. These results indicate that although translocation of OmpF-Lpp obligatory requires Δ μ˜ H+, the latter two proteins can be translocated in not only a Δ μ˜ H+-dependent manner but also a Δ μ˜ H+-independent manner.