Conductance of the SecY protein translocation channel

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 SecY complex in bacteria and archaea, opens across the membrane to enable hydrophilic polypeptide segments to cross the lipid bilayer. It also allows hydrophobic segments to pass through the channel walls into the lipid phase. In bacteria, the SecY channel collaborates with the ribosome during co- translational protein transport and with the cytosolic ATPase SecA during post-translational translocation. Our previous experiments have shown how the resting channel prevents small molecules from crossing the membrane: the barrier is maintained by a short helix, the plug, which is located in the center of the pore in close proximity to the pore ring, a constriction in the middle of the membrane (Saparov et al., 2007). The plug must move out of the way to allow the channel to open. We now propose to study how the channel maintains the barrier for small molecules when it is in action, i.e. when it translocates a polypeptide chain. We will reconstitute the purified translocation channel into planar bilayers and add either the channel partners - ribosomes or SecA - on their own, or generate translocation intermediates with a translocating polypeptide chain in the channel. We will then use electrophysiological measurements to determine conductivity and opening time of the channel. We will also perform fluorescence correlation spectroscopy to determine the concentration of unoccupied translocons, as well as translocons bound to ribosomes or SecA. These experiments will show whether the channel partners change the opening probability of the channel on their own and whether a translocating chain prevents small molecules from passing through the channel. Our work will therefore give insight into the mechanism by which the channel allows proteins to cross it, while maintaining the barrier for small molecules.

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. As ribosomal synthesis of membrane proteins progresses, the nascent chain must immediately enter the SecYEG channel because exposure to the aqueous environment would lead to denaturation. The signal which triggers SecYEG opening was thus far unknown. We were now able to show that ribosome binding to the resting SecYEG channel conveys that signal. The probability that the purified and reconstituted SecYEG channel opens upon ribosome docking is close to unity. To determine this probability, we developed a new assay based on a combination of single molecule fluorescence and single channel conductivity measurements. 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. We were now able to show that the channel does not exclude positively charged ions as falsely assumed up till now. Instead, the ion pathway closes in response to membrane voltage, once a translocation intermediate has been stalled. Insertion of numerous membrane proteins requires the SecYEG complex to interact with another protein, called insertase YidC. Our electrophysiological studies (performed in collaboration with H.G. Koch, Freiburg) revealed that YidC reduces SecYEGs channel diameter, by conceivably inserting itself into the lateral gate, i.e. the exit pathway for membrane proteins. Thus we obtained first mechanistic insight about how the nascent chain may be handed over from one protein to another to ensure proper folding in the membrane interior. We also studied the mechanism which governs the opening of the lateral gate. In collaboration with N. Bondar (Berlin) and S. White (Irvine), we identified a network of hydrogen bonds which were disrupted upon mutation of a single amino acid. As a result, the gate opens spontaneously, and the membrane barrier to ions is no longer maintained.Encouraged by the break-through in the understanding of the SecYEG YidC interaction, we also studied the purified YidC insertase (in collaboration with H.G. Koch, Freiburg) when reconstituted alone (i.e. without SecYEG). It was quite a surprise when the YidC insertase turned out to be a membrane channel that, very similar to SecYEG, opens upon ribosome binding to provide a pathway for the nascent chain to insert into the membrane during synthesis. Based on measurements of the unitary ion conductance, we concluded that channel diameter is smaller than that provided by SecYEG.