MEMBRANE INSERTION OF TAIL-ANCHORED PROTEINS IN Escherichia coli

• Main Author: Siti Shofiyah, Sofi
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/34996
• Institution: Institut Teknologi Bandung

Insertion of membrane proteins generally occurs co-translationally while the proteins are being synthesized in cytosol. In Escherichia coli, this process is generally mediated by the Sec translocase, a multimeric membrane protein complex that consist of membrane embedded SecYEG channel and the peripheral ATPase SecA. Membrane proteins are targeted to the translocase as ribosome nascent chains by the single recognition particle (SRP), which recognizes transmembrane segments (TMSs) when they emerge from the ribosomal polypeptide exit site. Some membrane proteins follow a different pathway and insert with the aid of the membrane protein YidC. In some membrane proteins the first TMS is localized at the extreme C-terminus, termed tail-anchored (TA) proteins. The location of the TMS has consequences for the biogenesis of TA proteins. Since the TMS only emerges from the ribosome after termination of protein synthesis, TA proteins cannot follow the general co- translational membrane targeting and insertion pathway, their biogenesis has recently attracted great interest. TA proteins are found in all kingdoms of life and have important roles in eukaryotes, such as SNARE proteins that operate during vesicular transport, as translocation machinery, regulate apoptosis, and mitochondrial morphology. TA proteins have been studied more extensively in eukaryotes rather than in prokaryotes. It is still unclear how these proteins targeted and inserted to the prokaryotic membrane. The aim of this study is to elucidate the biogenesis of TA proteins in the Gram-negative bacterium Escherichia coli. Based on in silico screening of the E. coli proteome for proteins categorized as TA proteins ElaB, a small 11 kDa protein of unknown function was chosen as a model. Prediction of the TMS with TMHMM software showed that the TMS of ElaB is formed in residues 80-99 followed by only two arginine residues. Two constructions of plasmid containing elaB gene was made both with and without N-terminal STREP-tag. ElaB containing an N-terminal STREP-tag was overexpressed in E. coli BL21(DE3) and analyzed with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. Membrane extraction and proteinase K protection assays confirmed that ElaB is an inner membrane protein and showed that its N-terminal domain is located in the cytoplasm. In vitro transcription-translation and membrane insertion of ElaB were performed in the presence of the indicated amounts of IMVs or (proteo)liposomes and the analyzed by autoradiography. Insertion of in vitro synthesized ElaB into inner membrane vesicles was stimulated by overexpression of SecYEG, but independent of YidC. The effect of proton motive force (PMF) on ElaB insertion was studied in the presence of IMVs supplemented with nigericin and valinomycin and showed to be depending on PMF. To study the role of SRP in the targeting of ElaB, assays were performed in the presence of FFh depleted lysates and showed that targeting of ElaB is SRP independent.