Sec translocon is the major machinery for protein translocation in E.coli including SecYEG, SecA and other Sec proteins. It is generally assumed that during translocation process, SecYEG serves as a protein-conducting channel and transports the protein across membranes by using SecA ATPase as driving force. However, previous work suggested that protein translocation can occur without SecYEG. In order to understand the role of SecA in this SecYEG-independent process, we use voltage clamp recording as a tool to study the ionic activity of SecA-dependent protein-conducting channel. In a major deviation from the conventional view, we found that SecA alone is sufficient to promote the channel activity with liposomes made of E.coli phospholipids in both whole cell recording in the oocytes and in the single channel recording with patch clamp. The activity is strictly dependent on the presence of functional SecA, including those from different species of bacteria. However, this SecA-alone dependent channel activity is less efficient compared to the membranes containing SecYEG. Furthermore, the channel activity loses the signal peptide specificity. Addition of purified SecYEG restores the signal peptide specificity as well as the efficiency. This channel activity is more sensitive to SecA-specific inhibitors compared with membranes containing wild-type SecYEG but is less sensitive to membranes containing suppressor proteins. This is the first time it has been shown that SecA binds to lipid low-affinity site and functions as a protein-conducting channel.
To further characterize the structural roles of SecA as the core of the channel, we use several SecA variants to reconstitute with liposomes to determine the domains involved in forming functional channels. Using deletion truncated domains of 901 residues SecA and liposomes in the oocytes recordings, we identify two critical SecA domains for the formation of pore channel activity: with phospholipids alone, and for interacting with SecYEG to gain higher activity. These data provide fundamental understanding for the SecA-dependent protein –conducting channels. Our findings also suggest the possible evolution process on the protein translocation pathways from prokaryotes through eukaryotes.
| Identifer | oai:union.ndltd.org:GEORGIA/oai:digitalarchive.gsu.edu:biology_diss-1108 |
| Date | 28 October 2011 |
| Creators | Hsieh, Ying-Hsin |
| Publisher | Digital Archive @ GSU |
| Source Sets | Georgia State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Biology Dissertations |
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