Targeted Actin Disassembly by Mical and Cofilin

Abstract

cell membrane and accumulate F-to toxic levels. One mechanism developed to mitigate this problem is the Fluc family of fluoride channels. These channels, extremely selective for fluoride over chloride and other anions, allow passive draining of fluoride down to sub-toxic levels. Recent structural work on two homologues of these proteins has demonstrated some very unusual characteristics. Like the small multidrug resistance transporter EmrE, Fluc is a dual-topology membrane protein, where the functional channel is an antiparallel homodimer made up of two Fluc molecules inserted into the membrane in opposite orientations. Surprisingly, structures of the Fluc proteins do not show a single, clear pore through the center of the dimer. Rather, there appears to be two independent ion pathways through the channel, with residues from both protein chains contributing to both pathways. We are currently using a combination of mutagenesis, electrophysiology, and crystallography to attempt to clearly delineate the pathways of ion conduction through the channel, and try to determine conclusively whether Fluc has a ''double-barreled'', parallel channel architecture. To that end, we have produced a functional Fluc concatomer, with both members of the antiparallel dimer contained in a single protein chain, linked by an additional transmembrane helix to maintain the antiparallel structure. This construct has allowed us to mutagenically degrade each pore independently, and thus demonstrate the presence of two independent pores through this small channel. 1747-PlatVoltage Sensitivity of the Bacterial Protein Translocation Channel The heterotrimeric protein translocation channel SecYEG enables (i) soluble proteins to cross the inner membrane and (ii) hydrophobic proteins to enter the membrane interior. It contains an aqueous pore that, in its resting state, is sealed by a ring of six hydrophobic residues and a half helix, termed the plug [1]. Signal sequence binding or ribosome binding both dislocate the plug and break the ring, thereby opening the channel to ions [2]. The membrane barrier to ions is preserved, since physiological values of the transmembrane potential close the channel by a yet unknown mechanism [3]. Here we demonstrate that this voltage sensitivity does not depend on the ligand. To be precise, the open time decreases together with a decrease in voltage for SecYEG channels that are bound to (i) signal peptides, (ii) translocation intermediates (proOmpA) and the motor protein SecA, (iii) a ribosome-nascent chain (FtsQ) complex or (iv) empty ribosomes.The observations were made with planar lipid bilayers that contained the purified and reconstituted SecYEG complex. They indicate that the voltage sensor must be part of the SecYEG channel. In our search for the sensor, we mutated charged residues, deleted the plug and performed various cross-link experiments, the outcome of which will be discussed.