In vivo analysis of an essential archaeal signal recognition particle in its native host

Abstract

The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.

FIG. 1.

Nucleotide sequence alignment of the 7S RNA homologs from H. volcanii, Halobacterium sp. strain NRC-1, and A. fulgidus.

FIG. 2.

Neighbor-joining tree generated by the alignment of seven conserved domains of SRP54 for representative organisms of the three domains of life. Internal nodes are labeled with the corresponding bootstrap confidence level (BCL), based on 100 bootstrap replicates of the alignment. Bootstrap confidence levels of <60% are not shown. Scale bar represents 0.1 amino acid substitution per site.

FIG. 3.

Construction and analysis of an H. volcanii hv54h-knockout strain. The chromosomal (chr) copy of hv54h was replaced with hv54h interrupted by the H. hispanica mevinolin resistance gene (mevR) in H. volcanii strain WRHv-6c (harboring the hv54h•6xhis expression plasmid pWR6-c), creating strain WRHv-6c/54KO (a). This strain and a control strain (WRHv-6c) were then examined as to whether they could be cured of pWR-6c in the absence of antibiotic (novobiocin) selection for the plasmid (b). At each indicated transfer, cells cultured in the absence of novobiocin were grown on RM supplemented with novobiocin. Error bars represent standard deviation. Student's paired t test was used to generate P values for each indicated transfer (T): P ≪ 0.0001 for T3, T9, T12, and T15; P = 0.012 for T6. nbR, novobiocin resistance gene.

FIG. 4.

Complementation of E. coli WAM113 depleted of Ffh. WAM-ffh and WAM-hv54h (Table 1) were grown without arabinose in the presence (a) or absence (b) of IPTG.

FIG. 5.

In vivo copurification of the H. volcanii SRP components. (a) Copurification with Hv54h•6xHis. The cytoplasmic fractions of strains WRHv-6c (Hv54h•6xHis) and WRHv-NP15 (control) were purified with Ni-NTA, and the cytoplasmic (cyto) and elution (elut) fractions were analyzed by Western blotting with antibodies against Hv54h and 6xHis (detecting Hv54h•6xHis), as well as by Northern blotting with a DNA probe for Hv7Sh. (b) Copurification with Hv19h•6xHis. The cytoplasmic fractions of WRHv-9a (Hv19h•6xHis) and WRHv-NP15 (control, not shown) were purified with Ni-NTA, and the elution fractions (E1 and E2) were analyzed by Western blotting with antibodies against 6xHis tag (detecting Hv19 h•6xHis) and Hv54h, as well as by Northern blotting with a DNA probe for Hv7Sh. The lower band observed in the anti-pentaHis Western blot is likely a degradation product of Hv19h•6xHis.