Voltage-dependence of the protein translocation channel (SecY)

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. The transporting channel, formed by the Sec61p complex in eukaryotes and the SecYEG complex in bacteria and archaea, opens across the membrane to enable secretory proteins to cross the lipid bilayer. It also allows membrane proteins to pass through the channel walls into the lipid phase. While facilitating transport of large protein across the membrane, the SecYEG complex must be closed to small ions. The SecYEG channel would otherwise provide a short-cut to the ionic solutions on both sides of the membrane and act like a battery with its two poles bridged; the bacterial cell would then run out of energy. Part of the rudimentary knowledge about the underlying mechanism is that transmembrane voltage closes SecYEGs gate to ions. We now aim at identifying this gate. That is, we want to find the voltage sensitive parts of the channel. These investigations will also help to understand how voltage facilitates protein transport by SecYEG. We propose to simultaneously visualize the movement of single polypetides and the openings of single channels. Site-directed charge adjustments of individual channel domains and the resulting alterations of protein and ion turnover numbers allow insight into the mechanism that utilizes the energy of the electric field for protein transport. That is, we will learn how exploiting the energy of the electric field tenfold decreases the expenses in chemical energy (stored in the form of ATP) for protein transport.

Protein synthesis takes place on ribosomes in the cytoplasm of a cell. All proteins that are secreted or incorporated into the plasma membrane use special transporters that facilitate (i) translocation through the membrane or (ii) membrane insertion. Conceivably, the Sec machinery represents the most important pathway. It can be found in all life forms. Its basic building block is formed in bacteria by a membrane channel consisting of three proteins, the so-called SecYEG complex. While transporting large proteins through the membrane, the SecYEG complex has to exclude small ions. Otherwise, the voltage difference between the cellular interior and the extracellular space would collapse - like the voltage of a battery when its poles are bridged. We disproved the previous assumption that parts of the SecYEG complex passively seal the channel by acting like a gasket. We have now shown that the seal requires energy input, i.e. that membrane voltage closes the channel by moving one of the gate posts of SecYEG. The gate post is part of a voltage sensor. The latter closes the channel after a hydrophobic protein segment has exited the lumen to enter the hydrophobic membrane interior. In contrast, a nascent hydrophilic protein segment does not completely depart from the channel and thus sterically hinders the return of the post to its original position. We have used the remaining ion flow as an indicator for the partition of the polypeptide chain from the lumen into the membrane interior. The measurements revealed that the equilibrium is reached only after a comparatively long time. A comparison with the much higher incorporation rates achieved by SecYEG complexes living cells, indicates the existence of a significant kinetic component. In other words, the decision to secrete the protein or to incorporate it into the membrane is not solely determined by protein hydrophobicity.