In vivo analysis of an essential archaeal signal recognition particle in its native host.
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.
Rose
R Wesley
RW
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Pohlschröder
Mechthild
M
D019843:Archaeal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D019622:Haloferax volcanii / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019642:RNA, Archaeal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020733:RNA, Small Cytoplasmic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D018271:Signal Recognition Particle / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
C115990:7SL RNA; D019843:Archaeal Proteins; D019642:RNA, Archaeal; D020733:RNA, Small Cytoplasmic; D018271:Signal Recognition Particle