Study of the five Rickettsia prowazekii proteins annotated as ATP/ADP translocases (Tlc):: Only Tlc1 transports ATP/ADP, while Tlc4 and Tlc5 transport other ribonucleotides
Study of the five Rickettsia prowazekii proteins annotated as ATP/ADP translocases (Tlc):: Only Tlc1 transports ATP/ADP, while Tlc4 and Tlc5 transport other ribonucleotides
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DOI：
10.1128/jb.00371-06
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发表时间：
2006-09-01
影响因子：
3.2
通讯作者：
Winkler, Herbert H.
中科院分区：
文献类型：
--
作者：
Audia, Jonathon P.;Winkler, Herbert H.
专性胞浆内致病菌普鲁泽立克次体依赖于真核宿主细胞细胞质中许多必需化合物的运输，而不是从头合成，这一进化结果无疑与这个充满代谢物的生态位的专性生长有关。立克次体转运系统研究的范例是ATP/ADP转位酶Tlc1，它将细菌ADP交换为宿主细胞ATP作为能量来源，而不是作为腺苷酸的来源。有趣的是，R. prowazekii基因组编码了四个开放阅读框，这些阅读框与已被充分表征的ATP/ADP转位酶Tlc1高度同源。因此，通过注释，R. prowazekii基因组共编码5个ATP/ADP转位：Tlc1、Tlc2、Tlc3、Tlc4和Tlc5。我们通过定量逆转录酶PCR证实，在L-929细胞中生长的proproazekii中有5个tlc同源基因对应的mrna表达，在大肠杆菌中也有异源蛋白表达，表明tlc基因在进化过程中都不是假基因。然而，我们通过在大肠杆菌中的异源表达证明，只有Tiel作为ATP/ADP转运蛋白。对核苷酸和核苷的调查已经确定Tlc4运输CTP、UTP和GDP。有趣的是，虽然GTP不通过Tlc4运输，但它是CTP和UTP摄取的抑制剂，并且表现出与GDP相似的K-i。此外，我们证明了Tlc5运输GTP和GDP。我们假设Tlc4和Tlc5的主要功能是维持立克次体核酸生物合成的细胞内核苷酸池，而不像Tlc1那样为细胞提供三磷酸核苷作为能量来源。虽然在大肠杆菌中观察到Tlc2和Tlc3的异源表达，但我们无法确定这些蛋白的底物。
The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tiel functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a K-i similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins.