Substrate-driven assembly of a translocon for multipass membrane proteins - Nature Chaperones HEAD TOPICS

Substrate-driven assembly of a translocon for multipass membrane proteins - Nature

10/22/2022 10:07:00 PM

Nature research paper Substrate-driven assembly of a translocon for multipass membrane proteins

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Nature research paper Substrate-driven assembly of a translocon for multipass membrane proteins Biochemical reconstitution and functional analysis reveal how newly synthesized multipass membrane proteins dynamically remodel the translocon to facilitate their successful biogenesis. doxycycline followed by transfection of single guide RNA targetingCCDC47−1hygromycin B for 2 weeks.and lysed in three volumes of hypotonic homogenization buffer (10 mM HEPES-KOH pH 7.5, 10 mM KOAc, 1 mM MgClfor 3 min at 4 °C and the supernatant was collected. The pellet was resuspended in 5 ml of insertion buffer (50 mM HEPES-KOH pH 7.5, 250 mM sucrose, 250 mM KOAc, 10 mM MgCl for 10 min at 4 °C. The supernatant was discarded, and the resulting membrane pellet was resuspended in insertion buffer (approximately 1 ml for about four plates). Microsomes (1-ml aliquots) were treated for 10 min at 37 °C with 4,000 U micrococcal nuclease (NEB), 2 U RNase-Free DNase (Promega), 1 mM CaCl Read more:
nature » Mechanism of an intramembrane chaperone for multipass membrane proteins - Nature How Much Protein You Should Eat Post-Workout + Best Protein Sources 16 RD-Approved High-Protein Breakfasts (That'll Keep You Full Until Lunch) How to smuggle a drug into cells: add a lipid ‘tail’

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This Roast Chicken with Hot Honey recipe gets its flavor from cardamom, raw honey, and orange peel. Get the recipe from Food & Wine. Read more >> Mechanism of an intramembrane chaperone for multipass membrane proteins - NatureBiochemical and structural analysis of intermediates during multipass membrane protein biogenesis showed how an intramembrane chaperone guides nascent membrane proteins to a semi-enclosed lipid-filled cavity where they are inserted and folded correctly. How Much Protein You Should Eat Post-Workout + Best Protein SourcesHow much protein should you eat after a workout? 16 RD-Approved High-Protein Breakfasts (That'll Keep You Full Until Lunch)Incorporating protein into your morning meal is an easy way to fuel your body for the busy day ahead, and the benefits for muscle gains, healthspan, and beyond are impressive. How to smuggle a drug into cells: add a lipid ‘tail’Molecules equipped with a lipid streamer of just the right length can wriggle through a cell’s defensive membrane. I think the authorities would probably spot that. Triassic sauropodomorph eggshell might not be soft - NatureMatters arising: Triassic sauropodomorph eggshell might not be soft 'This is progress': Environmental nonprofit sets out to restore Florida's declining oyster habitatsFlorida's Gulf Coast has been in rough shape for oysters. Decades of decline in oyster reefs — 85% globally, over the last two centuries — coupled with the 2010 oil spill in the Gulf of Mexico left the once-rich oyster beds sparse. Raise our taxes so we can eat oysters. No thanks cbs propaganda left wing news. .is solely for illustration purposes as a means of simultaneously displaying the best regions contributed by each map., gives her opinion on how much protein you need post-workout and where to get it.5 , to name a few," says Cording. An STT3A -knockout cell line was generated similarly. Briefly, Cas9 expression was induced by addition of 10 ng ml −1 doxycycline followed by transfection of single guide RNA targeting STT3A (5′-TCGACATTCGGAATGTCTGT-3′). b , Fourier shell correlation (FSC) curve for the Rho-2TMD reference map illustrating an overall resolution of 3. Cells were grown for 48 h, followed by single-cell sorting into 96-well plates for clonal isolation. If you meet your overarching daily protein needs , you don't necessarily need to pack in a protein-heavy meal after your workout. Clones were verified by both western blot and genomic sequencing. c Extended Data Fig. Stable cell lines expressing N-terminally Flag-tagged TMCO1 and nicalin in the corresponding knockout background were described previously ." Advertisement This ad is displayed using third party content and we do not control its accessibility features. A stable cell line expressing N-terminally Flag-tagged CCDC47 was generated similarly. a, b , Two views from the plane of the membrane of the Rho-2TMD reference map, low-pass filtered to 7 Å resolution, fitted with the model."As you get older, the blood flow to the muscles increases post-training, and there's. Briefly, a CCDC47 -knockout cell line was transfected with a modified pEGFP-n1 plasmid (Addgene) encoding N-terminally Flag-tagged CCDC47 (tag inserted after the signal peptide), under the control of a CMV promoter. Cells were transfected using TransIT-293 (Mirus) and selected in 0. The detergent micelle and ribosome are omitted for clarity.7 mg ml −1 G418 (Invitrogen) for 2 weeks, changing the medium every 3 days. After selection, cells were maintained in medium supplemented with 0. The clipping plane is within the membrane close to the ER lumen." Finally, don't get too hung up on counting every last gram. 3 mg ml −1 G418. Expression was verified by western blot. d , Overview of the low-pass filtered reference map in the context of the detergent micelle and ribosome. Stably integrated doxycycline-inducible ASGR1 and AGTR2 reporter lines for flow cytometry analysis were described previously . Other reporter lines were generated similarly. The blue line shows a low contour level at which lumenal density and the complete micelle is visible. Briefly, pcDNA5-based reporter constructs were co-transfected with pOG44 into Flp-In T-Rex 293 cells with TransIT-293, according to the manufacturer’s protocol (Invitrogen), and cells were selected in 100 μg ml −1 hygromycin B for 2 weeks. Advertisement This ad is displayed using third party content and we do not control its accessibility features. Preparation of rough microsomes HEK293 cells were grown to about 80% confluency in 15-cm dishes, washed once with 5 ml ice-cold PBS (per plate) and collected by scraping in 2 × 5 ml of PBS. The ribosome and micelle are in transparent grey. Cells were collected by centrifugation for 5 min at 500 g and lysed in three volumes of hypotonic homogenization buffer (10 mM HEPES-KOH pH 7.5, 10 mM KOAc, 1 mM MgCl 2 ) for 15 min on ice, with gentle agitation every few minutes. The position of the nascent chain density and the mouth of the ribosome exit tunnel are indicated. Cells were then homogenized by 15 strokes (up and down) in a chilled dounce tissue grinder. Sucrose was added to a final concentration of 250 mM and mixed gently. The top shows overviews before and after clipping, with dashed boxes indicating the regions shown at higher magnification below. Nuclei and cell debris were removed by centrifugation at 700 g for 3 min at 4 °C and the supernatant was collected. The pellet was resuspended in 5 ml of insertion buffer (50 mM HEPES-KOH pH 7. 4 AlphaFold2 predictions of the PAT, Sec61, GEL and BOS complexes.5, 250 mM sucrose, 250 mM KOAc, 10 mM MgCl 2 ) and centrifuged again. The pooled supernatant fractions were centrifuged at 10,000 g for 10 min at 4 °C. For all complexes, predicted alignment error (PAE) matrices, structural models coloured by the predicted Local Distance Difference Test (pLDDT) and the final models fitted into respective densities are shown. The supernatant was discarded, and the resulting membrane pellet was resuspended in insertion buffer (approximately 1 ml for about four plates). Microsomes (1-ml aliquots) were treated for 10 min at 37 °C with 4,000 U micrococcal nuclease (NEB), 2 U RNase-Free DNase (Promega), 1 mM CaCl 2 and 0. a , Human PAT complex, comprised of CCDC47 and Asterix.5 mM phenylmethylsulfonyl fluoride (PMSF), followed by quenching with 2 mM EGTA. Microsomes were pelleted at 10,000 g for 10 min at 4 °C and resuspended in 1 ml insertion buffer, 40 U SUPERaseIn and 0. ) filtered by local resolution.1 mM EGTA, followed by centrifugation at 10,000 g for 10 min at 4 °C. The supernatant was discarded, and the membrane pellet was resuspended in insertion buffer supplemented with 50 U SUPERaseIn (per four plates). The final model is fitted into the respective density taken from the Rho-2TMD reference map filtered by local resolution. The preparation was finally adjusted with insertion buffer to an absorbance at 260 nm ( A 260nm ) of about 50, and 50-µl aliquots were flash frozen in liquid nitrogen and stored at −80 °C for further use. Interaction analysis in stably integrated cells Microsomes from wild-type cells and cells encoding Flag-tagged versions of TMCO1, CCDC47 or Nicalin were prepared as above, except that the micrococcal nuclease digestion was performed with 10,000 U of micrococcal nuclease (NEB), 3 U DNAse (Promega), 1 mM CaCl 2 and 0. The final model (which omitted NOMO because it was not visualised in the map) is fitted into the respective density taken from the TMEM147 sub-classified map (submap 1 in Extended Data Fig.6 mM PMSF, and incubated at room temperature for 20 min before quenching with 2.5 mM EGTA. The density for Nicalin is also shown at a very low threshold in transparent white to visualise the lumenal domain density. Microsomes (1 ml at A 260nm = 50) were solubilized in insertion buffer supplemented with 2. 5% digitonin and 1× protease inhibitor cocktail (Roche, 11836170001) for 45 min on ice and then diluted twice with 150 mM KOAc insertion buffer. The final model is fitted into the respective density taken from the Rho-4TMD map filtered by local resolution. Digitonin-solubilized microsomes were cleared by centrifugation at 12,500 g for 15 min at 4 °C. The cleared supernatant ( A 260nm of about 3. 5 Characterisation of PAT complex reconstitution in SP cells.5) was immunoprecipitated in batch format using 50 µl M2 Flag affinity beads (Sigma, A2220) and gentle agitation overnight at 4 °C. Flow-through was removed and beads were washed three times with eight column volumes of insertion buffer supplemented with 0. Asterix KO cells contain residual levels of endogenous CCDC47. 4% digitonin. Bound material was eluted twice, for 30 min on ice, with two column volumes of 200 mM KOAc insertion buffer supplemented with 0. When substrate RNCs are subsequently introduced, their interaction with the PAT complex can be tested.5 mg ml −1 Flag peptide (ApexBio, A6001) and 0.4% digitonin. The cells were then isolated by centrifugation and analysed by protease protection to check Asterix topology (left) and interaction with CCDC47 by co-immunoprecipitation (IP; right). The eluate was collected using a pre-equilibrated spin filter column (Thermo Fisher, 69725). Ribosome-free and ribosome-bound fractions were obtained by pelleting the eluate through a 300-µl sucrose cushion (50 mM HEPES pH 7. The right panel shows the total in vitro translated products (lane 1), the sedimented SP cells (lane 2), native IP with CCDC47 antibody (lane 3) or native IP using control antibody (lane 4).4, 10 mM MgCl 2 , 150 mM KCl, 500 mM sucrose and 0.4% digitonin) at 355,000 g for 1 h at 4 °C in a TLA120. The cells were isolated and incubated with 35 S-labelled Rho RNCs truncated at 70 residues beyond TMD1.1 rotor. In vitro transcription and translation In vitro transcription reactions utilized PCR-based templates containing an SP6 promoter, and were carried out at 40 °C for 1 h (ref. The only cysteine of the substrate is located in the first TMD. ). Unless otherwise noted, reactions contained 5–10 ng μl −1 PCR product, 40 mM HEPES pH 7. For comparison, the sample containing BPA at M50 of Asterix was UV-irradiated, illustrating that photo-crosslinks between Asterix and substrate can also be visualised (see also Fig.6, 6 mM MgCl 2 , 2 mM spermidine, 10 mM dithiothreitol, 500 μM ATP, 500 μM UTP, 500 μM CTP, 100 μM GTP, 0.5 mM m7G(5′)ppp(5′)G RNA Cap, 0. As expected, the efficiency of photo-crosslinking is lower than chemical crosslinking.4 U μl −1 SUPERaseIn and 0. 4 U μl −1 SP6 RNA Polymerase. 35 S-labelled Asterix variants with BPA at the indicated positions were reconstituted into Asterix KO SP cells as in panel c. IVT reactions were performed using a RRL system (Green Hectares). Translation reactions contained 20% (v/v) of the unpurified transcription reaction, 33% (v/v) haemin- and micrococcal nuclease-treated RRL, 0. e , Non-radioactive asterix variants with BPA at indicated positions were reconstituted into Asterix KO SP cells as in panel c.2 μCi μl −1 .