Köster W, Gudmundsdottir A, Lundrigan M D, Seiffert A, Kadner R J
Department of Microbiology, School of Medicine, University of Virginia, Charlottesville 22908.
J Bacteriol. 1991 Sep;173(18):5639-47. doi: 10.1128/jb.173.18.5639-5647.1991.
The Escherichia coli btuB product is an outer membrane protein that mediates the TonB-coupled active transport of cobalamins and the uptake of the E colicins and bacteriophage BF23. The roles of various segments of the BtuB protein in its function or cellular localization were investigated by analysis of several genetic constructs. Hybrid proteins in which various lengths from the amino terminus of BtuB were linked to alkaline phosphatase (btuB::phoA genes) were all secreted across the cytoplasmic membrane. The BtuB-PhoA proteins that carried up to 327 amino acids of BtuB appeared to reside in the periplasmic space, whereas hybrid proteins containing at least 399 amino acids of BtuB were associated with the outer membrane. Eleven in-frame internal deletion mutations that spanned more than half of the mature sequence were prepared by combining appropriate restriction fragments from btuB variants with 6-bp linker insertions. None of the deleted proteins was able to complement any BtuB functions, and only three of them were detectable in the outer membrane, suggesting that most of the deletions affected sequences needed for stable association with the outer membrane. Duplications covering the same portions of BtuB were prepared in the same manner. All of these partial duplication variants complemented all BtuB functions, although some gave substantially reduced levels of activity. These proteins were found in the outer membrane, although some were subject to proteolytic cleavage within or near the duplicated segment. These results indicate that the insertion of BtuB into the outer membrane requires the presence of several regions of teh BtuB protein and that the presence of extra or redundant segments of the protein can be tolerated during its insertion and function.
大肠杆菌的btuB产物是一种外膜蛋白，它介导钴胺素的TonB偶联主动运输以及大肠杆菌素和噬菌体BF23的摄取。通过分析几种基因构建体，研究了BtuB蛋白不同区段在其功能或细胞定位中的作用。将来自BtuB氨基末端不同长度的片段与碱性磷酸酶相连的杂合蛋白（btuB::phoA基因）均分泌穿过细胞质膜。携带多达327个BtuB氨基酸的BtuB-PhoA蛋白似乎位于周质空间，而含有至少399个BtuB氨基酸的杂合蛋白与外膜相关。通过将来自btuB变体的合适限制性片段与6碱基对接头插入片段组合，制备了跨越成熟序列一半以上的11个读框内内部缺失突变体。没有一个缺失蛋白能够补充任何BtuB功能，并且其中只有三个在外膜中可检测到，这表明大多数缺失影响了与外膜稳定结合所需的序列。以同样的方式制备了覆盖BtuB相同部分的重复片段。所有这些部分重复变体都补充了所有BtuB功能，尽管有些变体的活性水平大幅降低。这些蛋白在外膜中被发现，尽管有些在重复片段内或附近受到蛋白水解切割。这些结果表明，BtuB插入外膜需要BtuB蛋白的几个区域的存在，并且在其插入和功能过程中，该蛋白额外或冗余片段的存在是可以被容忍的。
