Sahin-Tóth M, Lawrence M C, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5421-5. doi: 10.1073/pnas.91.12.5421.
An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance. Introduction of the mutation Glu-325-->Cys into either the first or the second half of the dimer results in a 50% decrease in activity, whereas introduction of the mutation into both halves of the dimer abolishes transport. Lactose transport by permease dimer is completely inactivated by N-ethylmaleimide; however, 40-45% activity is retained after N-ethylmaleimide treatment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues. The observations demonstrate that both halves of the fusion protein are equally active and suggest that each half may function independently. To test the possibility that oligomerization between dimers might account for the findings, a permease dimer was constructed that contains two different deletion mutants that complement functionally when expressed as untethered molecules. Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer interact or that there is an oligomeric interaction between dimers. The approach is consistent with the contention that the functional unit of lactose permease is a monomer.
一种含有两个串联乳糖通透酶分子的工程融合蛋白（通透酶二聚体）表现出高转运活性，并用于测试负显性现象。将Glu-325→Cys突变引入二聚体的前半部分或后半部分会导致活性降低50%，而将该突变引入二聚体的两个半部分则会使转运完全丧失。通透酶二聚体的乳糖转运被N-乙基马来酰亚胺完全灭活；然而，当二聚体的前半部分或后半部分被不含半胱氨酸残基的突变体取代时，N-乙基马来酰亚胺处理后仍保留40-45%的活性。这些观察结果表明融合蛋白的两个半部分活性相同，并表明每个半部分可能独立发挥功能。为了测试二聚体之间的寡聚化是否可以解释这些发现，构建了一种通透酶二聚体，它包含两个不同的缺失突变体，当作为非连接分子表达时，它们在功能上互补。由于这种构建体在任何程度上都不催化乳糖转运，二聚体的两个半部分相互作用或二聚体之间存在寡聚相互作用的可能性不大。该方法与乳糖通透酶的功能单位是单体这一观点一致。
