Jung H, Rübenhagen R, Tebbe S, Leifker K, Tholema N, Quick M, Schmid R
Universität Osnabrück, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie, Barbarastrabetae 11, D-49069 Osnabrück, Germany.
J Biol Chem. 1998 Oct 9;273(41):26400-7. doi: 10.1074/jbc.273.41.26400.
Hydropathy profile analysis of the amino acid sequence of the Na+/proline transporter of Escherichia coli (PutP) suggests that the protein consists of 12 transmembrane domains (TMs) which are connected by hydrophilic loops (Nakao, T., Yamato, I., and Anraku, Y. (1987) Mol. Gen. Genet. 208, 70-75). We have tested this prediction by applying a gene fusion approach in combination with a Cys accessibility analysis and site-specific proteolysis. Characterization of a series of PutP-alkaline phosphatase (PhoA) and PutP-beta-galactosidase (LacZ) hybrid proteins yields a reciprocal activity pattern of the reporter proteins that is in agreement with the topology of TMs III to XII of the 12-helix model. Placement of the PutP-PhoA and PutP-LacZ junction sites closer to the N terminus does not yield conclusive results. As a prerequisite for further topology studies, a functional PutP molecule devoid of all five native Cys residues (Cys-free PutP) is generated. Subsequently, amino acids in Cys-free PutP are replaced individually with Cys, and the accessibility of the sulfhydryl groups is analyzed. Surprisingly, Cys residues placed close to the N terminus of PutP (Ile-3 --> Cys, Thr-5 --> Cys) or into putative TM II (Ser-71 --> Cys, Glu-75 --> Cys) are highly accessible to membrane permeant and impermeant thiol reagents in intact cells. In contrast, Cys at the C terminus (Ser-502 --> Cys) reacts only with the membrane permeant but not with the impermeant reagent in intact cells. These results contradict the 12-helix motif and indicate a periplasmic location of the N terminus whereas the C terminus faces the cytoplasm. In addition, a transporter with Cys in place of Leu-37 (putative periplasmic loop (pL2) shows the same accessibility pattern as the Cys at the C terminus. Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orientation, is readily cleaved by the endoproteinase AspN before Asp-33 (pL2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp-356 (cL9). These results suggest a cytosolic location of Asp-33 and Leu-37, thereby implying the formation of an additional TM formed by amino acids of pL2. Based on these observations, a new secondary structure model is proposed according to which the protein consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytoplasm. The 13-helix structure is discussed as a common topological motif for all members of the Na+/solute cotransporter family.
对大肠杆菌的Na⁺/脯氨酸转运蛋白（PutP）氨基酸序列进行的亲水性分析表明，该蛋白由12个跨膜结构域（TMs）组成，这些结构域由亲水环连接（中尾彻、大和一、荒乐洋，（1987年）《分子与普通遗传学》208卷，70 - 75页）。我们通过将基因融合方法与半胱氨酸可及性分析和位点特异性蛋白酶解相结合来验证这一预测。对一系列PutP - 碱性磷酸酶（PhoA）和PutP - β - 半乳糖苷酶（LacZ）杂合蛋白的表征产生了报告蛋白的反向活性模式，这与12螺旋模型中TMs III至XII的拓扑结构一致。将PutP - PhoA和PutP - LacZ连接位点放置得更靠近N端并未得出确凿结果。作为进一步拓扑研究的前提，构建了一个不含所有五个天然半胱氨酸残基的功能性PutP分子（无半胱氨酸PutP）。随后，将无半胱氨酸PutP中的氨基酸逐个替换为半胱氨酸，并分析巯基的可及性。令人惊讶的是，位于PutP N端附近的半胱氨酸残基（Ile - 3→Cys，Thr - 5→Cys）或放入假定的TM II中的半胱氨酸残基（Ser - 71→Cys，Glu - 75→Cys）在完整细胞中对膜通透性和非通透性硫醇试剂高度可及。相反，C端的半胱氨酸（Ser - 502→Cys）在完整细胞中仅与膜通透性试剂反应，而不与非通透性试剂反应。这些结果与12螺旋基序相矛盾，表明N端位于周质，而C端面向细胞质。此外，用半胱氨酸取代Leu - 37的转运蛋白（假定的周质环（pL2））显示出与C端半胱氨酸相同的可及性模式。此外，以内外翻转方向纯化并重构到蛋白脂质体中的PutP，在Asp - 33（pL2）、Asp - 112（假定的细胞质环（cL3））、Asp - 262（cL7）和Asp - 356（cL9）之前很容易被内肽酶AspN切割。这些结果表明Asp - 33和Leu - 37位于细胞质中，从而意味着由pL2的氨基酸形成了一个额外的TM。基于这些观察结果，提出了一种新的二级结构模型，根据该模型，该蛋白由13个TMs组成，N端在外侧，C端面向细胞质。13螺旋结构被讨论为Na⁺/溶质共转运蛋白家族所有成员的一种常见拓扑基序。
