Timothy L. Yahr

Tim Yahr's Curriculum Vitae Email:Tim.Yahr@Dartmouth.edu

My research interests can be broadly defined as understanding the mechanisms by which bacterial proteins are transported into or across lipid bilayers. Specifically, I am interested in the type III translocation systems which have been identified in nearly all significant Gram-negative pathogens (ie., Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, Escherichia coli, Yersinia sp.). Type III translocation systems are highly complex and specialized structures which facilitate the delivery of toxins from the cytosol of adherent bacteria directly to the cytoplasm of eukaryotic host cells. Since the bulk of the research efforts to date have focused on the biological effects of the translocated toxins on eukaryotic host cells, almost nothing is known about the biochemical basis of of type III translocation. My long term research goal is to investigate and understand the mechanisms of type III translocation.

In Bill Wickner´s laboratory I have been studying preprotein translocase. This enzyme catalyzes the translocation of signal peptide bearing preproteins from the cytosol to the periplasm or facilitates the translocation and integration of polytopic integral membrane proteins into the inner membrane. One current hypothesis in the field is that the active translocase is a tetramer of SecYEG heterotrimeric complexes.

To biochemically identify the translocationally active translocase, I identified detergent solubilization conditions which allowed for the solubilization of the translocase with an arrested translocase intermediate. Using these solubilization conditions I developed an assay for the detection of oligomeric SecYEG by incorporating an epitope tag on one of the translocase components. By co-expressing the epitope tagged subunit along with the wildtype subunit, we reasoned that both the tagged and untagged subunits would assemble into the oligomeric complex. Under all conditions examined however, immunoprecipitations with the epitope tag failed to precipitate the untagged subunit suggesting that active form of translocase is simply a monomer of SecYEG.

More recently, I initiated work on the twin-arginine translocation (Tat) pathway of E. coli. This Sec-independent translocation pathway transports cofactor (ie., Fe-S clusters, molybdopterin guanine dinucleotide, polynuclear copper) containing proteins with characteristic twin-arginine signal sequence motifs into or across the inner membrane. Acquisition of the co-factor has been demonstrated to be a prerequisite for translocation which implies that proteins are secreted in at least partially folded conformations. My work with the Tat pathway has focused on the development of an in vitro translocation assay and significant progress has been made towards this goal. Having this assay working and in hand, I will be able to begin defining the minimal components of the translocase, energy requirements, and the mechanism of substrate recognition.