Supplementary Materials Supplemental Materials supp_23_3_464__index. SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins. INTRODUCTION Transport of proteins from your cytoplasm into the cytoplasmic membrane is an essential process in bacterial physiology. To facilitate protein insertion into lipid membranes, efficient transport systems have evolved, which include the membrane-embedded SecYEG translocon and the YidC insertase (Pohlschr?der and other Gram-negative bacteria is connected via a long periplasmic loop to an additional N-terminal TM (S??f but can be functionally replaced by the mitochondrial Oxa1 (van Bloois results in a global switch in cell physiology (Price membrane. RESULTS Functional reconstitution of SecYEG and YidC into proteoliposomes Identifying the determinants that route proteins into the YidC-only or into the SecYEG/YidC pathway by in vivo experiments has been complicated because the conditional depletion of SecY or YidC induces a multifaceted response that includes the up-regulation of proteases and chaperones (Baars strain in which SecY and YidC can be inactivated individually or together (Pop lipids supplemented with 5% diacylglycerol Ldb2 (DAG), which has been shown to reduce nonphysiological spontaneous membrane protein insertion (Nishiyama phospholipids supplemented with 5% DAG. Purified SecYEG or YidC were reconstituted into liposomes and pelleted by centrifugation. The pellet was resuspended and adjusted to a final concentration of 0.4 mg/ml SecY, 0.4 mg/ml YidC, or 0.4 mg/ml SecY + order Sitagliptin phosphate 0.4 mg/ml YidC. One aliquot (4 l) was loaded onto a 15% SDS gel and Coomassie stained. Pure liposomes served as control. (B) The proteoliposomes (2 l) shown in A had been separated on SDSCPAGE and blotted onto a nitrocellulose membrane. The membrane was cut into two parts, as well as the top part was decorated with -YidC antibodies and the lower part with -SecY antibodies. order Sitagliptin phosphate (C) One aliquot of the purified in vitro TT system utilized for in vitro protein synthesis was probed with the indicated antibodies after Western blotting. Purified FtsY (0.7 g), Ffh (0.2 g), SecA (1.5 g), SecY (0.8 g), and order Sitagliptin phosphate YidC (0.8 g) served as settings. The purified in vitro transcription/translation system (TT system) used in this study does not consist of significant amounts of FtsY, Ffh, SecA, SecY, or YidC (Koch inner membrane vesicles (INVs), we observed a membrane-protected fragment (MPF) of 19 kDa after proteinase K (PK) treatment (Number 2B) that was not observed in the absence of INVs. A similar protease-protected fragment was also observed in in vivo pulse-chase experiments (Yi INVs was further confirmed by alkaline carbonate extraction, a method that is routinely used to differentiate between membrane-inserted and soluble proteins (Fujiki inner membrane vesicles (WT INV; 2 mg/ml). After synthesis, one-fourth of the reaction was precipitated with TCA, and the remainder was first treated with 0.5 mg/ml PK for 30 min at 25C and then TCA precipitated. Full-size TatC (TatC) and the TatC-MPF are indicated. Note that wild-type INV contains adequate amounts of SRP and FtsY (Koch ribosomes The observation that YidC307-RNCs can be cross-linked to YidC shows a cotranslational connection, which is also supported from the stimulatory effect of SRP and the recent modeling of RNCs onto a YidC dimer (Kohler 70S ribosomes were incubated with or without detergent-solubilized, purified, His-tagged YidC or SecY and centrifuged through a sucrose cushioning. The amount of YidC or SecY in the supernatant and pellet fraction was then immunodetected using antibodies against their respective.