FTSH, A MEMBRANE-BOUND ATPASE, FORMS A COMPLEX IN THE CYTOPLASMIC MEMBRANE OF ESCHERICHIA-COLI
FTSH, A MEMBRANE-BOUND ATPASE, FORMS A COMPLEX IN THE CYTOPLASMIC MEMBRANE OF ESCHERICHIA-COLI
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DOI：
10.1074/jbc.270.40.23485
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发表时间：
1995-10-06
影响因子：
4.8
通讯作者：
ITO, K
中科院分区：
文献类型：
--
作者：
AKIYAMA, Y;YOSHIHISA, T;ITO, K
大肠杆菌的FtsH （HflB）蛋白通过两个位于n端的跨膜片段整合到膜中，而其大的细胞质结构域与AAA家族的atp酶同源。先前对显性阴性FtsH突变体的研究提出了FtsH在多聚体状态下起作用的可能性，我们发现在尺寸排除色谱中，从单体结构中洗脱的FtsH的分子量比预期的要大。用交联剂二硫比酯（琥珀酰丙酸）处理膜或其洗涤剂提取物，产生FtsH的交联产物。为了解剖可能的FtsH复合物，我们构建了在其C端具有C -Myc表位的FtsH衍生物（FtsH- his (6)-Myc）。当用交联剂处理由过量产生FtsH- his (6)-Myc和正常FtsH的细胞制备的膜时，完整的FtsH和没有标记的FtsH- his (6)-Myc的体外降解产物与标记的FtsH蛋白交联，共免疫沉淀实验证实了FtsH分子之间的相互作用。为了确定这种相互作用所需或足够的FtsH区域，我们在FtsH和EnvZ之间构建了嵌合蛋白。我们发现，只有具有ftsH衍生的膜锚定结构域和envz衍生的细胞质结构域的ftsH - envz杂交蛋白引起显性ftsH表型，并与ftsH交联。我们认为，ftsH的n端跨膜区域直接介导了ftsH亚基之间的相互作用。
The FtsH (HflB) protein of Escherichia coli is inte grated into the membrane with two N-terminally located transmembrane segments, while its large cytoplasmic domain is homologous to the AAA family of ATPases, The previous studies on dominant negative ftsH mutants raised a possibility that FtsH functions in multimeric states, We found that FtsH was eluted at fractions corresponding to a larger molecular weight than expected from monomeric structure in size-exclusion chromatography, Moreover, treatment of membranes or their detergent extracts with a cross-linker, dithiobis(succinimidyl propionate), yielded cross linked products of FtsH, To dissect possible FtsH complex, we constructed an FtsH derivative with c-Myc epitope at its C terminus (FtsH-His(6)-Myc). When membranes prepared from cells in which FtsH-His(6)-Myc was overproduced together with the normal FtsH were treated with the cross-linker, intact FtsH and in vitro degradation products of FtsH-His(6)-Myc without the tag were cross linked with the tagged FtsH protein, Co-immunoprecipitation experiments confirmed the interaction between the FtsH molecules, To identify regions of FtsH required or sufficient for this interaction, we constructed chimeric proteins between FtsH and EnvZ, a protein with a similar topological arrangement, by exchanging their corresponding domains, We found that only the FtsH-EnvZ hybrid protein with an FtsH-derived membrane anchoring domain and an EnvZ-derived cytoplasmic domain caused a dominant ftsH phenotype and was cross-linked with FtsH, We suggest that the N-terminal transmembrane region of FtsH mediates directly the interaction between the FtsH subunits.