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

All living organisms can be classified into three domains: the Eukarya, the Bacteria, and the Archaea (45). While the archaea are morphologically similar to the bacteria, at the molecular level they exhibit a combination of unique as well as bacterial and eukaryotic characteristics (18, 31). Recent whole-genome analyses of numerous archaea have expanded our understanding of this domain and provided insight into phylogenetic relationships, putative metabolic pathways, and cellular processes (3, 14, 37, 38). 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). However, in addition to homologs of the universally conserved Sec components, archaea possess a mixture of homologs of bacterial and eukaryotic translocation components (29, 31). This observation, as well as the fact that homologs of translocation ATPases from Bacteria or Eukarya are lacking (e.g., SecA and Kar2p/BiP, respectively), suggest unique features of the archaeal protein translocation pathway (29, 31). Studying the significance of the similarities and differences among the Sec pathways of Archaea, Bacteria, and Eukarya will further our understanding of translocation in all the domains. 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). In contrast, the composition of the eukaryotic SRP receptor (SR), a component of the ER membrane that docks the SRP nascent chain to the membrane, shows less similarity to its archaeal homolog (29). The eukaryotic SR is a heterodimer composed of Srα and a membrane protein, Srβ, that anchors the Srα subunit to the ER membrane. In contrast, bacteria and archaea seem only to contain a homolog of the Srα subunit, called FtsY (29, 32). Despite the lack of a prokaryotic Srβ homolog, FtsY is found in both cytoplasmic and membrane-associated pools in the bacterial and archaeal species examined to date (6, 20, 25). FtsY is a member of the universally conserved SRP subfamily of GTPases whose members are characterized by the presence of a four alpha-helical bundle (termed the N domain) and immediately followed by the catalytic GTPase region (termed the G domain) (8). Both of these domains are involved in recognition of the universally conserved protein component of the SRP, SRP54 (termed Ffh in bacteria), and play critical roles in SRP-dependent protein translocation (9, 12). Many, though not all, examples of FtsY include an additional amino-terminal domain, named the “A” domain (9, 11, 12, 15). This name originated from the largely acidic character of the first example of FtsY to be described, that from Escherichia coli. Although not universal, many FtsY examples also have acidic A domains, including FtsY homologs from the haloarchaea (A. Haddad and M. Pohlschroder, unpublished observations). In the absence of obvious membrane spanning regions in FtsY, it had been thought that the amino-terminal A domain mediated the interaction of FtsY with the membrane (7, 23, 24). However, this view recently has been challenged by several observations. First, sequence alignments reveal that in contrast to the strongly conserved N and G domains, the A domain is poorly conserved (29). Moreover, several bacterial and archaeal FtsY homologs lack an A domain (9, 11, 12). Second, the essential role of the A domain has been reevaluated in light of recent observations in E. coli, demonstrating that expression of FtsY lacking an A domain functionally complements disruption of the full-length protein (11). The amino-terminally truncated construct also associated with the E. coli cytoplasmic membrane, albeit less efficiently than full-length FtsY (11). 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). We wished to further characterize the archaeal FtsY homolog in vivo. In the present study, we have employed a recently developed gene knockout strategy to demonstrate in vivo that the H. volcanii FtsY homolog is crucial for growth (2). Furthermore, we show that a haloarchaeal FtsY construct lacking the amino-terminal A domain is able to complement a chromosomal ftsY deletion and that in both strains the SRP is targeted to the membrane. Thus, although the archaeal SRP-dependent pathway shares similarities with that of both eukaryotes and bacteria, the archaeal FtsY appears in vivo to be more similar to the E. coli FtsY.