Mecky Pohlschröder,
Ph. D.

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

To remain viable, an organism must translocate a subset of its proteins into or through hydrophobic cellular membranes. The proteins translocated through these membranes play a variety of invaluable functional role in critical cellular processes, which include nutrient acquisition, toxin secretion, signal transduction and the formation of extracytoplasmic protein complexes, such as pili and flagella.

The pathways used by cells to facilitate protein translocation include the Sec system, which transports proteins in an unfolded conformation and is thought to be the major translocation pathway used by most organisms, and the twin-arginine translocation (Tat) system, which transports unfolded proteins. These translocation pathways have been characterized in bacteria and in eukaryotes, and have recently been described in archaea.

Our lab focuses on identifying and characterizing the molecular components in these translocation pathways in archaea. Characterization of these pathways in archaea will allow us to: 1) define general principles for each type of translocation pathway; 2) determine the bases for the preferential transport of specific proteins via the Sec or Tat pathway; and 3) based on the similarities and differences the pathways in archaea, bacteria and eukaryotes, determine evolutionary relationships.

We study these translocation pathways in the model system Hfx. volcanii, a haloarcheon that is amenable to genetic and biochemical manipulation and analyses. In addition to addressing the aims stated above, by elucidating the biological processes that are dependent on the substrates translocated by these pathways in haloarchaea, which thrive in salt conditions approaching saturation, we may be able to identify specific adaptations made by this species to high salt concentrations. We are complementing our in vivo studies of the mechanisms underlying protein translocation with computational in silico analyses of pathway components and the substrates of these pathways. As part of this work we have developed several signal peptide identification programs (signalfind.org).

The haloarchaeal Sec-pathway
Most proteins pass through the endoplasmic reticular membranes of eukaryotes and the cytoplasmic membranes of bacteria via a proteinaceous pore known as the Sec translocon. While the core components of these pores are evolutionarily conserved in bacteria and eukaryotes, the specific functions of most of these components are not well understood. Interestingly, the archaeal Sec pathway contains a combination of bacterial and eukaryotic Sec component homologs. Curiously, no archaeal homolog of a translocation ATPase has yet been identified. Using a combination of genetic, biochemical and in silico approaches, we have been identifying and characterizing Hfx. volcanii homologs of known Sec components and we are also attempting to identify as yet unknown Sec pathway components, including an archaeal Sec translocation ATPase. (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
The haloarchaeal Tat pathway: Many archaeal species possess unique adaptations that allow survival in extreme environments. In haloarchaea, in silico and in vivo evidence obtained by our lab suggests that, while the Sec pathway is crucial for haloarchaeal growth, haloarchaea route most secreted proteins through the Tat pathway, www.sas.upenn.edu/~pohlschr/, which may be an adaptation to the high salt concentrations found in their natural environments. This unique characteristic makes studying the haloarchaeal Tat pathway particularly useful since a wide range of substrates is available for use in characterizing the functions of known Tat components as well as for screens and selections that can be used to identify additional Tat components. Moreover, revealing the significance of haloarchaea-specific Tat pathway characteristics and comparing these characteristics to those of non-haloarchaeal pathways may provide important information concerning the efficient use of the Tat pathway and lead to a better understanding of pathway mechanisms in general. Using this system, we can also address crucial problems such as: 1) determining the dynamics of pore assembly; 2) discovering how proteins having drastically differing sizes can be translocated while maintaining the essential semipermeability of the membrane; and 3) identifying the selective pressures that target substrates to the Tat or Sec pathway.

Finally, it should be noted that eukaryotic Tat pathway components have only been identified in chloroplasts. Since Tat mutants of pathogenic Escherichia coli and Pseudomonas aeroginosa, along with Tat mutants of other bacterial pathogens, are attenuated for virulence, certain components of this pathway may be attractive drug targets. (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.

 

Archaeal type IV pilus-like structures
Although bacterial flagella and archaeal flagella have similar functions, that is, they are responsible for swimming motility in these prokaryotes, the synthesis and structures of these protein complexes are very different. In fact, the synthesis of archaeal flagella closely resembles that of bacterial type IV pili and the structural subunits of these complexes, which are secreted via the Sec pathway, are also related. Since Hfx. volcanii are capable of biological processes that in bacteria require the involvement of type IV pili, based on sequence data generated for archaeal flagellin, we developed a software program that allowed us to identify several operons in the Hfx. volcanii genome that encode pilin-like proteins, as well as similar operons in many other archaeal genomes. We have now determined the temporal expression patterns for these Hfx. volcanii operons and we are currently optimizing conditions for their expression. We have also generated several mutant strains and we are attempting to determine the roles these pilin-like proteins play in surface adhesion, autoaggregation, twitching motility and conjugation, all processes that in bacteria require functional type IV pili (Fig. 3).

Figure 3. Haloferax volcanii wild-type swimming motility ( right) in comparison to that of two motility mutants recently identified in an insertional mutagenesis screen.