Biochemical and genetic evidence for three transmembrane domains in the class I holin, λ S
Biochemical and genetic evidence for three transmembrane domains in the class I holin, λ S
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DOI：
10.1074/jbc.275.2.769
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发表时间：
2000-01-14
影响因子：
4.8
通讯作者：
Young, R
中科院分区：
文献类型：
--
作者：
Gründling, A;Bläsi, U;Young, R
I类霍林蛋白的原型基因S编码3个潜在的跨膜结构域，其107个密码子编码3个潜在的跨膜结构域，而II类基因原型21 S仅编码71个密码子，编码2个跨膜结构域。许多Holin基因，包括Lambda S和21岁的S，在N端都有“双启动”调控基序，这表明I类和II类Holin具有相同的拓扑结构。21 S的一级结构强烈地暗示了一种双视位的螺旋-发夹拓扑结构，N和C末端位于细胞质一侧。然而，具有N端信号序列的波长S嵌合体显示出Lep依赖的功能，表明S的N端结构域需要输出。这里的信号序列嵌合体对错义改变A52V很敏感，错义改变阻碍了正常的S功能。此外，利用一组具有单半胱氨酸取代的S变异体在离体膜上进行的半胱氨酸修饰研究表明，S蛋白3个可能的跨膜结构域的核心位置受到保护。此外，预测的细胞质和周质环中具有单半胱氨酸取代的S蛋白分别在倒膜囊泡和整个细胞中被更有效地标记。这些数据构成了Holin S-lambda具有三个跨膜结构域的直接证据，并表明尽管在调控和功能上具有同源性，但I类和II类Holin具有不同的拓扑结构。
lambda S, the prototype class I holin gene, encodes three potential transmembrane domains in its 107 codons, whereas 21 S, the class II prototype spans only 71 codons and encodes two transmembrane domains. Many holin genes, including lambda S and 21 S, have the "dual-start" regulatory motif at the N terminus, suggesting that class I and II holins have the same topology. The primary structure of 21 S strongly suggests a bitopic "helical-hairpin" topology, with N and C termini on the cytoplasmic side of the membrane. However, lambda S chimeras with an N-terminal signal sequence show Lep-dependent function, indicating that the N-terminal domain of S requires export. Here the signal sequence chimera is shown to be sensitive to the missense change A52V, which blocks normal S function. Moreover, cysteine-modification studies in isolated membranes using a collection of S variants with single-cysteine substitutions show that the positions in the core of the 3 putative transmembrane domains of lambda S are protected. Also, S proteins with single-cysteine substitutions in the predicted cytoplasmic and periplasmic loops are more efficiently labeled in inverted membrane vesicles and whole cells, respectively. These data constitute direct evidence that the holin S-lambda has three transmembrane domains and indicate that class I and class II holins have different topologies, despite regulatory and functional homology.