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

Abstract

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 Tlc1 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.

FIG. 1.

Assay of ATP transport by the R. prowazekii Tlc homologues. The tlc1 through tlc5 homologues were cloned into the pET-11a expression vector and introduced into the C41 strain of E. coli by electroporation. Cultures were grown, induced, and assayed for transport of ATP as described in Materials and Methods. ATP was added at a concentration of 0.05 mM (with 1 to 2 μCi/ml of [α-³²P]ATP), and uptake reactions were initiated by the addition of cells. Symbols: Tlc1, filled squares; Tlc2, filled diamonds; Tlc3, filled triangles; Tlc4, open squares; and Tlc5, open circles.

FIG. 2.

Assay of NXP transport by Tlc4 (A) and Tlc5 (B). The tlc4 and tlc5 genes were cloned into the pET-11a expression vector and introduced into the C41 strain of E. coli by electroporation. Cultures were grown, induced, and assayed for the transport of ATP, CTP, UTP, GTP, and GDP as described in Materials and Methods. All NXPs were added at a concentration of 1 mM (with 1 to 2 μCi/ml of the appropriate α-³²P-labeled nucleotide phosphate), and uptake reactions were initiated by the addition of cells. Symbols: ATP, open squares; CTP, filled squares; UTP, filled diamonds; GTP, open triangles; and GDP, filled triangles.

FIG. 3.

Effect of increasing concentrations of GTP and GDP on the transport of CTP by Tlc4. The tlc4 gene was cloned into the pET-11a expression vector and introduced into the C41 strain of E. coli by electroporation. Cultures were grown, induced, and assayed for transport as described in Materials and Methods. CTP was added as a substrate at a concentration of 0.2 mM (with 1 to 2 μCi/ml of [α³²P]CTP), and transport was assayed in the presence of increasing concentrations of GTP and GDP (0, 1, 2, 4, and 6 mM). Symbols: GTP, filled squares; GDP, filled diamonds. The dotted line represents the theoretical curve for an inhibitor with a K_i of 1 mM. GXP, GDP or GTP.