Mecky Pohlschröder,
Ph. D.

Associate Professor of Biology
Ph.D., University of Massachusetts, Amherst, 1994

protein translocation in archaea

The transport of proteins across hydrophobic membranes is a process essential to the survival of all organisms. Several mechanisms have evolved to facilitate this transport, including 1) the Sec system, which is thought to transport the majority of unfolded proteins; and 2) the twin arginine translocation (Tat) system, which transports folded proteins. While Sec translocation occurs in bacteria, archaea, and eukaryotes, Tat translocation has only been observed in prokaryotes and chloroplasts.
Our long-term goal is to understand these mechanisms in archaea. By studying these processes in organisms of this domain, which share characteristics with both bacteria and eukaryotes, we hope to 1) illuminate general principles involved in each type of transport; 2) understand the relative costs and advantages of each; and 3) determine how each process has been evolutionarily adapted to suit the needs of these particular organisms. We characterize these pathways in Haloferax volcanii, an archaeon that is amenable to modern molecular and biochemical techniques, and can be easily cultured under standard laboratory conditions.

The haloarchaeal Sec-pathway
Most proteins pass through the endoplasmic reticular membrane of eukaryotes and the cytoplasmic membrane of bacteria via a proteinaceous pore, the Sec-translocon. While the core components of the pore are evolutionarily conserved, many sec-components are distinct in bacteria and eukaryotes and the functions of most of these proteins are not well understood. Genome analyses of completely sequenced archaea suggest that organisms of this domain of life contain a combination of bacterial and eukaryotic Sec component homologs, and lack a homolog of the bacterial and eukaryotic translocation ATPases (Fig.1).

Figure 1. Sec machinery components in representatives of bacteria (E. coli), archaea (H. volcanii) and eukaryotes (S. cerevisiae). Sec substrates are translocated into or across hydrophobic membranes via the universally-conserved heterotrimeric Sec61 (SecYEG in bacteria) pore. Translocation through this protein-conducting channel requires distinct sets of additional Sec components in bacteria, archaea and eukaryotes. YidC and TRAM are only involved in the insertion of proteins into the bacterial cytoplasmic and the ER membrane, respectively. While ATP hydrolysis by SecA and Kar2p are involved in energizing Sec translocation in bacteria and eukaryotes, respectively, no archaeal translocation ATPases have been identified. cyt - cytoplasm.

Our in vivo analyses strongly suggest that the sec pathway is essential in organisms of this domain of life. Understanding how sec-substrates pass through the cytoplasmic membrane of H. volcanii will not only provide important information about the evolutionary relationships of these organisms, but also raise new questions about the mechanism of protein translocation in general.

The haloarchaeal Tat pathway
In vivo and in silico data from our lab suggest that halophilic archaea, including H. volcanii, which thrive in environments with salt concentrations approaching saturation, employ the Tat pathway for the majority of their proteins. This differs significantly from the non-haloarchaea, which only use this pathway for a subset of their proteins and is likely to be an adaptation to the high salt conditions by allowing cytoplasmic folding of secreted proteins prior to their secretion (http://www.sas.upenn.edu/~pohlschr/).

Our observation is not only interesting in light of the evolutionary adaptation of these organisms to their environment. Studying the Tat pathway in H. volcanii will help us in learning more about this still poorly understood pathway in general. Crucial questions like, “Which components target proteins to the pore?" "What are the dynamics of pore assembly?" and "What are the exact functions of previously identified Tat components?" have yet to be answered (Fig.2).

Figure 2. Tat components and model of Tat secretory mechanism. (A) Typcial structure of Tat machinery components in bacteria and archaea. The post-amphipathic helical C-terminus for TatA and TatB has been excluded for visual simplicity. (B) Model of Tat substrate translocation in E. coli. Tat substrates (oval) obtain tertiary structure in the cytoplasm and are targeted to the membrane TatBC complex in an unknown manner. Once bound to substrate, the TatBC complex interacts with a multimeric TatA ring in a DpH-dependent manner. The plugged inactive TatA ring likely alters to an active unplugged confirmation upon engaging substrate. There is insufficient data describing points of protein interactions, and the depicted points of interaction between proteins is not meant to be completely accurate.

Furthermore, as mentioned above, eukaryotic homologs of Tat components have only been identified in chloroplasts. Considering the fact that Tat mutants in pathogenic Escherichia coli and Pseudomonas aeroginosa have been shown to be attenuated for virulence in an animal model, components of this pathway may be identified as drug targets (http://www.sas.upenn.edu/~pohlschr/tatprok.html).