Voltage-dependence of the protein translocation channel (SecY)
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Many proteins need to be transported across the membrane of the endoplasmic reticulum (ER) or across the bacterial plasma membrane during or after their biosynthesis. A protein complex that is conserved throughout all kingdoms of life ?the Sec61p complex in eukaryotes and the SecYEG complex in bacteria and archaea ? represents a major transporting pathway. It acts as a transmembrane pore for secretory proteins, and allows hydrophobic segments to pass through its lateral gates into the lipid phase. In bacteria, the SecYEG channel collaborates with the cytosolic ATPase SecA during post-translational translocation. Our previous experiments have shown that physiological values of membrane potential are required to prevent small molecules from crossing the activated channel. We now intend to identify the voltage-sensitive element, i.e. to study key steps in SecYEG?s voltage-driven conformational change. Since the rate of protein translocation is also known to be voltage-dependent, we will examine whether the voltage-sensing element is the same for the facilitation of translocation and the enforcement of channel closure. We will also investigate co-translational translocation to learn whether the voltage-sensitive mechanism for maintaining the membrane barrier to small molecules is the same as in post-translational translocation, i.e. whether it remains unaltered when SecYEG is not interacting with SecA but rather with the ribosome nascent chain complex. We will reconstitute the purified translocation channel into horizontal planar bilayers with stalled translocation intermediates in the channel. We will use mutational analysis to modify conserved charged residues to determine SecY?s dwell time in the open state during electrophysiological measurements....