Membrane binding of SRP pathway components in the halophilic archaea Haloferax volcanii

Abstract

Across evolution, the signal recognition particle pathway targets extra‐cytoplasmic proteins to membranous translocation sites. Whereas the pathway has been extensively studied in Eukarya and Bacteria, little is known of this system in Archaea. In the following, membrane association of FtsY, the prokaryal signal recognition particle receptor, and SRP54, a central component of the signal recognition particle, was addressed in the halophilic archaea Haloferax volcanii. Purified H. volcanii FtsY, the FtsY C‐terminal GTP‐binding domain (NG domain) or SRP54, were combined separately or in different combinations with H. volcanii inverted membrane vesicles and examined by gradient floatation to differentiate between soluble and membrane‐bound protein. Such studies revealed that both FtsY and the FtsY NG domain bound to H. volcanii vesicles in a manner unaffected by proteolytic pretreatment of the membranes, implying that in Archaea, FtsY association is mediated through the membrane lipids. Indeed, membrane association of FtsY was also detected in intact H. volcanii cells. The contribution of the NG domain to FtsY binding in halophilic archaea may be considerable, given the low number of basic charges found at the start of the N‐terminal acidic domain of haloarchaeal FtsY proteins (the region of the protein thought to mediate FtsY–membrane association in Bacteria). Moreover, FtsY, but not the NG domain, was shown to mediate membrane association of H. volcanii SRP54, a protein that did not otherwise interact with the membrane.

Streptococcal viability and diminished stress tolerance in mutants lacking the signal recognition particle pathway or YidC2

“…The A domain has been reported as essential for proper targeting of membrane proteins (42); however, more recent in vivo analyses showed that, in E. coli, the N-terminal A-domain was not required for SRP-mediated protein targeting and membrane assembly (43). In contrast, in the halophilic Archaea Haloferax volcanii, the full-length FtsY is essential for the association of Ffh protein with the cell membrane (44). Therefore, the mode of action and requirement of the A domain appears to differ among species.…”

The Haloferax volcanii FtsY Homolog Is Critical for Haloarchaeal Growth but Does Not Require the A Domain

“…Furthermore, a more complete understanding of the Archaea has proven to have important implications for our understanding of cellular functions conserved in Eukarya and Bacteria (21,34). This has been demonstrated in studies examining archaeal protein translocation via the universally conserved Sec pathway, which is thought to be responsible for the translocation of the majority of proteins into and across the endoplasmic reticular (ER) or cytoplasmic membranes in eukaryotes and prokaryotes, respectively (19,35). Specifically, the notable achievement of the crystal structure of an archaeal Sec translocation pore, the first such structure obtained from any organism, has greatly contributed to the understanding of this universally conserved pore in all organisms (42).…”

“…Recent in vivo and in vitro analyses of the protein translocation mechanisms of Haloferax volcanii, a halophilic archaeon amenable to genetic and biochemical analyses, have complemented genomic and structural analyses of the archaea (19,35). The composition of the archaeal SRP is more similar to that of eukaryotes than the composition of bacterial SRP (10,29,35).…”

“…Finally, in vitro analysis of purified proteins interacting with H. volcanii inverted vesicles showed that both the full-length haloarchaeal FtsY homolog and a version of FtsY lacking the A domain were able to associate with these membrane preparations (19). Interestingly, additional in vitro experiments suggested that the A domain may be involved in recruiting the SRP to the haloarchaeal cytoplasmic membrane (19).…”

In the Archaea Haloferax volcanii, Membrane Protein Biogenesis and Protein Synthesis Rates Are Affected by Decreased Ribosomal Binding to the Translocon

“…Comprising an eukaryal-like SRP and a bacterial-like SRP receptor (20,21), the archaeal SRP pathway could serve to target translating ribosomes to the membrane, as in Eukarya and Bacteria. To date, archaeal SRP from several species has been reassembled from purified components (22)(23)(24) and has been isolated from H. volcanii (25), whereas binding of the archaeal SRP receptor to membrane vesicles has also been shown (26,27). Moreover, as in Eukarya (28,29) and Bacteria (30,31), recent in vitro studies have revealed that the translocon also serves as the membrane ribosomal receptor in Archaea (32).…”