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Damon Huber

Wednesday 19 October 2016 - Understanding the mechanism of Sec-dependent protein translocation from the inside out.

Sec-dependent protein translocation is a fundamental biological pathway, which is responsible for transporting proteins across the cytoplasmic membrane. However, although the mechanism of translocation across the membrane has been investigated extensively, the early steps in this process are only poorly understood. I will present three interconnected lines of research into the cytoplasmic steps in Sec-dependent protein translocation: First, it was recently discovered that the ATPase that drives translocation in bacteria, SecA, binds to ribosomes. This result was surprising because protein translocation in bacteria is generally uncoupled from protein synthesis (i.e. it is “posttranslational”). However, using a combination of genetic and biochemical approaches, we have found that SecA binds to nascent Sec substrate proteins as they emerge from the ribosome suggesting that substrate protein recognition is cotranslational. Second, we carried out a high-throughput genetic screen (Transposon Directed Insertion Site Sequencing—TraDIS) for mutants that are more sensitive to the SecA inhibitor sodium azide. Experiments following up the results of this screen suggest that SecA is allosterically regulated in response to changes in the environment. Finally, we have identified a truncation variant of SecA that has a much higher affinity for ribosomes. Studies investigating the structure of this variant have provided insight into the conformational changes between the active and autoinhibited forms of SecA. Taken together, our research suggests that the recognition of substrate proteins by SecA and their subsequent delivery to the membrane for translocation is complex and highly regulated.