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Dec 2019
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In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes
细菌膜蛋白在蛋白质脂质体中的体外整合   

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Abstract

It is important to experimentally determine how membrane proteins are integrated into biomembranes to unveil the roles of the integration factors, and to understand the functions and structures of membrane proteins. We have developed a reconstitution system for membrane protein integration in E. coli using purified factors, in which the integration reaction in vivo is highly reproducible. This system enabled not only analysis of membrane-embedded factors including glycolipid MPIase, but also elucidation of the detailed mechanisms underlying membrane protein integration. Using the system, the integration of membrane proteins can be evaluated in vitro through a protease-protection assay. We report here how to prepare (proteo)liposomes and to determine the activities of membrane protein integration.

Keywords: Membrane protein integration (膜蛋白整合), SecYEG (SecYEG), YidC (YidC), MPIase (膜蛋白整合酶), SRP (信号识别颗粒), Diacylglycerol (甘油二脂), Proteoliposomes (蛋白质脂质体)

Background

Membrane proteins and presecretory proteins, synthesized in the cytosol, are integrated into and translocated across biomembranes, respectively, to be localized at their destinations and to express their functions. Cells possess systems to integrate membrane proteins into and translocate presecretory proteins across biomembranes, these proteins being commonly conserved from bacteria to higher eukaryotes.

In E. coli, a model organism, some integration factors were identified through genetic approaches, these factors including translocon SecYEG (Newitt and Bernstein, 1998), and signal recognition particle (SRP) and its receptor SR (Ulbrandt et al., 1997), and membrane protein insertase/chaperone YidC (Samuelson et al., 2000). These genetic studies were complemented by biochemical approaches. The molecular mechanisms underlying membrane protein integration have been studied extensively using an in vitro system, in which the protein integration reactions were reproduced in test tubes.

Inverted membrane vesicles (INV) can be prepared by disrupting E. coli cells with a French press, followed by sucrose gradients (Alami et al., 2002). The outside surface of INV corresponds to the cytoplasmic face of the inner membrane. Therefore, presecretory proteins are translocated into the lumen of INV. Membrane proteins, synthesized outside of INV using the cell-free translation system, are integrated into INV. The roles of the above-mentioned factors have been analyzed using INV prepared from the respective mutant cells (Koch et al., 1999; Koch and Muller, 2000). However, since most of the factors are essential for cell growth, depletion of each of the factors causes unexpected pleiotropic effects. To exclude such secondary effects, a reconstitution system has been developed using purified factors. Proteoliposomes can be prepared by mixing membrane proteins, solubilized and purified in a detergent solution, and phospholipids, followed by removal of the detergent. The reconstituted proteoliposomes are essentially the same as INV and thus they can be directly used in the integration assay in vitro. On the other hand, disordered spontaneous integration of membrane proteins into liposomes formed of phospholipids is a serious problem, since such spontaneous integration is an in vitro artefact that does not reflect the reaction in vivo. We have solved this problem by introducing diacylglycerol (DAG) into the liposomes (Nishiyama et al., 2006; Kawashima et al., 2008). As a consequence, DAG blocked spontaneous integration at the physiological content, which enabled reconstitution of membrane protein integration. By using such a reconstitution system we identified a glycolipid named MPIase (Membrane Protein Integrase) as an essential factor for membrane protein integration (Nishiyama et al., 2006; Nishiyama et al., 2010). Based on the observation that MPIase catalyzes membrane protein integration (Nishiyama et al., 2010), we proposed that MPIase is a “glycolipozyme” (Nishiyama et al., 2012). We also determined the MPIase structure, in which a glycan chain composed of 9~11 repeats of a unit of three N-acetylated amino sugars, GlcNAc, ManNAcA and Fuc4NAc, is connected to DAG through a pyrophosphate linker (Nishiyama et al., 2012). Thus, membrane protein integration can be reproduced by introducing lipid components such as DAG and MPIase into reconstituted proteoliposomes.

Recently, we found another problem when reconstituting proteoliposomes. DAG is not only solubilized by a series of detergents such as dodecyl maltoside (DDM) and dodecyl phosphocholine (DPC), but also forms wax-like complexes with them (Sasaki et al., 2019). Since these wax-like complexes induce the spontaneous integration, the detergents must be removed completely from the reconstituted proteoliposomes. On the other hand, DDM and DPC are frequently used to purify the SecYEG complex (Collinson et al., 2001) and YidC (Stiegler et al., 2011; Welte et al., 2012). Therefore, the membrane components should be properly reconstituted without the formation of the unexpected complexes that induce spontaneous integration.

In this protocol, we report a method for reconstituting proteoliposomes containing SecYEG and YidC, but free of the complex of DAG and DDM/DPC. To achieve this, SecYEG and/or YidC were first reconstituted into DAG-free proteoliposomes, followed by membrane fusion with liposomes containing DAG and MPIase by freezing-thawing-sonication, which yielded unilamellar proteoliposomes (Sasaki et al., 2019). Such proteoliposomes are ready for the membrane integration assay. This assay system includes a Pure System, a reconstituted translation system, to in vitro synthesize substrate membrane proteins (Nishiyama et al., 2010; Shimizu et al., 2001). The integration activity was determined by analyzing the membrane protected fragments (MPF) (Koch et al., 1999). The MPFs generated upon protease digestion after the integration reaction reflect the membrane integration.

Materials and Reagents

  1. Plastic tubes (1.5 ml) (Greiner, catalog number: 616201 )
  2. Ultracentrifuge tubes (1.5 ml) (Beckman Coulter, catalog number: 357448 )
  3. Glass vials (2 ml) (AS ONE, catalog number: 9-852-01 )
  4. Dialysis tubes (Tokyo Garasu Kikai Co., Ltd, catalog number: 0 3272337 )
  5. E. coli polar lipid extract (phospholipids) (Avanti Polar Lipids, Inc., catalog number: 100600C )
  6. 1,2-Dioleoyl-sn-glycerol (DAG) (Merck, catalog number: D0138 )
  7. 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Dojindo Laboratories, catalog number: 342-01375 )
  8. Dithiothreitol (Wako, catalog number: 048-29224 )
  9. Chloroform, Guaranteed Reagent (Wako, catalog number: 038-02606 )
  10. Acetone, Guaranteed Reagent (Wako, catalog number: 012-00343 )
  11. n-Octyl-β-D-glucopyranoside (OG) (Dojindo Laboratory, catalog number: O001 )
  12. [35S]-EXPRESS Protein Labeling Mix (PerkinElmer Inc, catalog number: NEG072 )
  13. Pure System (Gene Frontier, catalog number: PF001-0.25 )
  14. Proteinase K (Roche, catalog number: 03 115 879 001 )
  15. Trichloroacetic acid, Guaranteed Reagent (Wako, catalog number: 204-02405 )
  16. Acrylamide, Electrophoresis grade (Wako, catalog number: 011-08015 )
  17. N,N'-Methylenebis(acrylamide), Electrophoresis grade (Wako, catalog number: 130-06031 )
  18. Sodium dodecyl sulfate (SDS), for Molecular Biology (Wako, catalog number: 194-13985 )
  19. 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris), Guaranteed Reagent (Wako, catalog number: 207-06275 )
  20. Na2HPO4, Guaranteed Reagent (Wako, catalog number: 197-02865 )
  21. NaH2PO4, Guaranteed Reagent (Wako, catalog number: 192-02815 )
  22. Glycine, Guaranteed Reagent (Wako, catalog number: 077-00735 )
  23. Bromophenol Blue (Wako, catalog number: 021-02911 )
  24. β-Mercaptoethanol (Wako, catalog number: 131-14572 )
  25. Glycerol, Guaranteed Reagent (Wako, catalog number: 075-00611 )
  26. Ffh, purified as described (Eisner et al., 2003)
  27. FtsY, purified as described (Koch et al., 1999)
  28. YidC, purified as described (Nishikawa et al., 2017)
  29. SecYEG, purified as described (Moser et al., 2013)
  30. MPIase, purified as described (Nishiyama et al., 2010 and 2012)
  31. TALON Metal Affinity Resin (Clontech, catalog number: 635503 )
  32. Imaging plates (GE, BAS IP MS 2025 E, catalog number: 28-9564-75 )
  33. Buffer A (see Recipes)
  34. OG stock (see Recipes)
  35. Sodium phosphate buffer (pH 7.2) (see Recipes)

Equipment

  1. Bath-type sonicator (Branson, model: Bransonic 12 )
  2. Freeze-dryer (EYELA, model: FD-80 )
  3. Vacuum pump (ULVAC, model: GCD-051X )
  4. Ultracentrifuge (Beckman Coulter, model: Optima TL )
  5. Rotor (Beckman Coulter, model: TLA-55 )
  6. Incubator (Eppendorf, model: Thermomixer Comfort )
  7. Phosphorimager (GE, model: Storm 820 )
  8. -80 °C freezer

Software

  1. ImageQuant software (GE Healthcare)

Procedure

  1. Preparation of proteoliposomes containing proteinaceous factors such as SecYEG and YidC
    1. Place the phospholipid solution (25 mg/ml in chloroform) in a glass vial. For one reconstitution sample, use 200 μg of phospholipids. Dry phospholipids up under a N2 gas stream until the bulk of chloroform is evaporated, and then place in a freeze-dryer under vacuum for 1 h.
    2. Suspend the phospholipids (200 µg/one reconstitution sample) in buffer A (50 mM HEPES-KOH, pH7.5, 1 mM dithiothreitol) at 10 mg/ml, followed by bath-sonication (about 10 s) to form liposomes until a slightly opaque suspension is obtained.
    3. Solubilize liposomes thus prepared in 1.5% [w/v] OG in buffer A by adding 15% [w/v] stock.
    4. Mix the solubilized phospholipids (200 µg) with SecYEG (~10 μg) and/or YidC (~10 μg) solubilized in 1.5% [w/v] OG. Adjust the volume to ~100 µl, keeping the OG concentration at 1.5% [w/v]. Incubate the mixture on ice for 20 min.
    5. Change the detergents used to solubilize SecYEG and YidC to OG, if they were solubilized in DDM or DPC. Apply the sample to a TALON column, and then wash with buffer containing 1.5% [w/v] OG, followed by elution with buffer containing 100 mM imidazole. In the case of SecYEG, add 40% [w/v] glycerol to the buffer. Keep the DDM/DPC concentration as low as possible.
    6. Dialyze the mixture against 500 ml of buffer A at 4 °C for at least 3 h to form proteoliposomes by removing OG. The proteoliposome suspension becomes a bit turbid.
    7. Add 0.9 ml of buffer A to the dialyzed sample, followed by recovery of proteoliposomes by centrifugation (170,000 x g, 1 h, 4 °C). Resuspend the pellets in 50 µl of buffer A.
    8. Make proteoliposomes unilamellar by freezing (in liquid nitrogen)-thawing (at room temperature)-sonication (~10 s) once. Make sure that the suspension is not heated when it is sonicated.
    9. Store frozen proteoliposomes at -80 °C, if necessary, before thawing and sonication.

  2. Preparation of liposomes containing DAG
    1. Mix phospholipids (1 mg) and DAG (0.05 mg), and dry the lipid mixture up as in Step A1.
    2. Suspend the dried residue in buffer A at 10 mg phospholipids/ml, followed by bath-sonication (~10 s).

  3. Preparation of liposomes containing DAG and MPIase
    1. Mix phospholipids and DAG in chloroform, and dry the mixture up, as described in Step B1. Typically, add DAG at 25% [w/w] to phospholipids.
    2. Dissolve the mixture in solvent C (chloroform/ethanol/water = 3/7/4), and then mix with MPIase dissolved in solvent C, typically at 25% [w/w] as to phospholipids.
    3. Evaporate solvent C under a N2 stream and then under vacuum.
    4. Suspend the dried residue in buffer A at 10 mg phospholipids/ml, followed by bath-sonication (~10 s).

  4. Fusion of proteoliposomes with MPIase/DAG-liposomes to incorporate MPIase and DAG
    1. Mix the proteoliposome suspension obtained in Procedure A with MPIase/DAG-liposomes (Procedure C), typically in the ratio of 4:1, which yields proteoliposomes containing 5% [w/w] DAG and 5% [w/w] MPIase.
    2. Repeat the cycle of freezing (in liquid nitrogen)-thawing (at room temperature)-sonication (~10 s) three times to allow liposome fusion. With this way, proteoliposomes containing SecYEG, YidC, and MPIase/DAG can be prepared.

  5. Reactions for (3L-)Pf3 coat integration (Figure 1)
    1. Translate in vitro the substrate proteins, Pf3 coat and the mutant 3L-Pf3 coat (Kiefer and Kuhn, 1999; Serek et al., 2004), using the Pure System, a reconstituted cell-free system for in vitro translation (Nishiyama et al., 2010; Shimizu et al., 2001). The reaction mixture of the Pure System (20 µl) comprises [35S] methionine (2.5~10 MBq/ml; ~200 nM), plasmid DNA (pT7-3L-Pf3 or pT7-Pf3 [Kawashima et al., 2008; Serek et al., 2004]; ~20 µg/ml), and (proteo)liposomes (0.4 mg phospholipids/ml) (Table 1). When a high level of synthesis is necessary, add cold methionine (0.3 mM) to the mixture. Under these conditions, a ~200-fold increase in the synthesis level is observed. The Pure System is commercially available from Gene Frontier, but adjust the buffer system customly as described (Nishiyama et al., 2010). To avoid aggregation of proteoliposomes, make sure that the Mg concentration is 9 mM or less, and that PEG and polyamines such as putrescine and spermidine are not contained in the reaction mixtures.
    2. Allow the translation/integration reaction at 37 °C for 30 min.
    3. Terminate the reaction by chilling on ice for 5 min. Withdraw 3 µl and 15 µl of the mixture (20 µl) into new tubes.
    4. Add 3 µl of trichloroacetic acid (10% [w/v]) to an aliquot (3 µl) to precipitate the synthesized proteins.
    5. Mix an equal volume of proteinase K (1 mg/ml) with another aliquot (15 µl), and then incubate at 25 °C for 25 min to generate MPF.
    6. After proteinase K digestion, add 30 µl of 10% [w/v] trichloroacetic acid, followed by incubation at 56 °C for 5 min to inactivate proteinase K. Incubate on ice for at least 10 min to precipitate MPF.
    7. Recover the precipitates (both Steps E4 and E6) by centrifugation (10,000 x g, for 5 min), and then wash them with 100% acetone. After centrifugation (10,000 x g, for 5 min), dry the precipitates up at room temperature for 20~30 min.
    8. Solubilize the precipitates in 10 µl of SDS sample loading buffer A, followed by boiling for 3 min. The SDS sample loading buffer A is composed of 25 mM sodium phosphate (pH 7.2), 2.5% [w/v] SDS, 25% [w/v] glycerol, 2.5% [w/v] β-mercaptoethanol, and 0.0125% [w/v] bromophenol blue.
    9. Apply samples onto SDS-gels containing 6 M urea as described in Nishiyama et al. (2006). The gel is composed of 12.5% [w/v] acrylamide-0.27% [w/v] N,N'-methylenebis(acrylamide) in 100 mM sodium phosphate buffer (pH 7.2) as described in Hussain et al. (1980).
    10. After electrophoresis, soak the gels in 10% [v/v] acetic acid for 10 min, and then dry them.
    11. Expose the gels to the imaging plates in a cassette overnight.

      Table 1. Composition of the Pure System



      Figure 1. Scheme of the assay for (3L-)Pf3 coat integration

  6. Reactions for MtlA (mannitol permease) integration
    1. Synthesize MtlA as in E, using pET-MtlA (Kuruma et al., 2005) and the Pure System. Add Ffh (50 µg/ml), FtsY (17 µg/ml), and cold methionine (30 µM) to the reaction mixture.
    2. After the translation/integration reaction at 37 °C for 30 min, add 5% [w/v] trichloroacetic acid to an aliquot (9 µl), and digest another aliquot (9 µl) with proteinase K as described in Steps E4-E5. Recover the precipitate as described in Step E5.
    3. Solubilize the dried precipitates in 10 µl of SDS sample loading buffer B, followed by boiling for 3 min. The SDS sample loading buffer B is composed of 62.5 mM Tris-HCl (pH 6.8), 3% [w/v] SDS, 10% [w/v] glycerol, 5.5% [w/v] β-mercaptoethanol, and 0.01% [w/v] bromophenol blue.
    4. Load samples onto SDS-gels as described (Laemmli, 1970). The gel is composed of 15% [w/v] acrylamide-0.4% [w/v] N,N'-methylenebis(acrylamide).
    5. After electrophoresis, soak gels in 10% [v/v] acetic acid for 10 min, and then dry them. Place the gels on the imaging plates.

Data analysis

Determination of the integration activity

  1. Detect radioactive bands of the synthesized substrates and MPF using a Phosphorimager.
  2. Determine the radioactivity of each band using the ImageQuant software (Figure 2). Subtract the background level manually with the software.
  3. Determine the integration activities by dividing the MPF level with that of the synthesized substrate (Figure 2, bottom of each gel image). Consider the numbers of methionine in substrates and MPF.


    Figure 2. Examples of the results of the integration assay. A. Pf3 coat integration depends on MPIase and is stimulated by YidC. B. Both MPIase and SecYEG are essential for MtlA integration. Moreover, YidC stimulates the MPIase/SecYEG-dependent integration of MtlA. The numbers of methionine (25 for MtlA and 18 for MtlA-MPF) were taken into account for activity determination. Images were taken from Sasaki et al., 2019 (Figures 3C and 3D).

Recipes

  1. Buffer A
    Dilute 1 M HEPES-KOH (pH 7.2) and 1 M dithiothreitol solutions to 50 mM and 1 mM, respectively, with ddH2O
  2. OG stock
    Dissolve OG in ddH2O at 15% [w/v], and store at 4 °C
  3. Sodium phosphate buffer (pH 7.2)
    1. Prepare 1 M NaH2PO4 and 1 M Na2HPO4 solutions
    2. Mix these, monitoring the pH, to yield 1 M sodium phosphate buffer (pH 7.2)
    3. Stock this solution at room temperature

Acknowledgments

This work was supported by Japan Society for the Promotion of Science Grants-in-Aid 18J21847 (to H. N.); 15KT0073, 16H01374, 16K15083, 17H02209 and 18KK0197 (to K. N.). This protocol was derived from our report (Sasaki et al., 2019).

Competing interests

We declare that we have no competing interests.

References

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简介

[摘要 ] 实验确定膜蛋白如何整合到生物膜中以揭示整合因子的作用,并了解膜蛋白的功能和结构非常重要。我们已经开发了一种重组系统,用于使用纯化因子在大肠杆菌中整合膜蛋白,其中体内的整合反应可高度重现。该系统不仅能够分析包括糖脂MPIase在内的膜嵌入因子,而且能够阐明膜蛋白整合背后的详细机制。使用该系统,可以在体外评估膜蛋白的整合 通过蛋白酶保护测定。我们在这里报告了如何准备(PROTEO )脂质体,并确定膜蛋白结合的活动。

[背景技术 [ 0002 ] 在胞质溶胶中合成的膜蛋白和分泌蛋白分别被整合到生物膜中并跨生物膜转运,以定位在它们的目的地并表达其功能。细胞拥有将膜蛋白整合到生物膜中并使其分泌的蛋白跨生物膜的系统,这些蛋白通常从细菌到高级真核生物都是保守的。

在一种模式生物大肠杆菌中,通过遗传方法鉴定了一些整合因子,这些因子包括SloSecYEG (Newitt和Bernstein,1998),信号识别颗粒(SRP)及其受体SR (Ulbrandt 等,1997)。,和膜蛋白插入酶/分子伴侣YidC (Samuelson et al。,2000)。这些基因研究得到了生化方法的补充。使用体外系统对膜蛋白整合的分子机制进行了广泛的研究,其中蛋白质整合反应在试管中进行。

倒膜囊泡(INV)可通过用French press 破坏大肠杆菌细胞,然后用蔗糖梯度制备(Alami 等,2002)。INV的外表面对应于内膜的细胞质面。因此,分泌蛋白被转移到INV腔中。使用无细胞翻译系统在INV外部合成的膜蛋白被整合到INV中。利用从各个突变细胞制备的INV已经分析了上述因素的作用(Koch 等,1999;Koch和Muller,2000)。但是,由于大多数因素对于细胞生长都是必不可少的,因此每种因素的消耗都会导致意想不到的多效性效应。为了排除这种次要作用,已经开发了使用纯化因子的重建系统。可以通过混合膜蛋白,在去污剂溶液中增溶和纯化以及磷脂,然后去除去污剂来制备蛋白脂质体。重组的蛋白脂质体与INV基本相同,因此可直接用于体外整合试验。另一方面,膜蛋白自发无序地自发整合到由磷脂形成的脂质体中是一个严重的问题,因为这样的自发整合是一种体外假象,不能反映体内的反应。我们通过将二酰基甘油(DAG)引入脂质体中来解决了这个问题(Nishiyama 等,2006;Kawashima 等,2008)。结果,DAG在生理含量上阻止了自发整合,这使得能够重建膜蛋白整合。通过使用这种重构系统,我们鉴定出了名为MPIase (膜蛋白我ntegr ASE )作为膜蛋白融合的一个重要因素(西山等人。2006年, ; 西山。等,2010)。基于MPIase催化膜蛋白整合的观察(Nishiyama 等,2010),我们提出MPIase是一种“糖脂酶” (Nishiyama 等,2012)。我们还确定了MPIase结构,其中由3个N- 乙酰化氨基糖GlcNAc,ManNAcA和Fuc4NAc 的一个单元的9〜11 个重复组成的聚糖链通过焦磷酸盐接头与DAG连接(Nishiyama 等,2012)。)。因此,可通过将脂质成分(例如DAG和MPIase)引入重构的蛋白脂质体中来复制膜蛋白整合。

最近,我们在重组蛋白脂质体时发现了另一个问题。DAG不仅被一系列去污剂如十二烷基麦芽糖苷(DDM)和十二烷基磷酸胆碱(DPC)溶解,还与它们形成蜡样复合物(Sasaki 等,2019)。由于这些蜡状复合物诱导自发整合,因此必须从重构的蛋白脂质体中完全去除去污剂。另一方面,DDM和DPC经常用于纯化SecYEG复合物(Collinson 等,2001)和YidC (Stiegler 等,2011;Welte 等,2012)。因此,应适当地重组膜成分,而不会形成诱导自发整合的意外复合物。

在该协议中,我们报告了一种重建包含SecYEG和YidC的蛋白脂质体的方法,但不含DAG和DDM / DPC的复合物。为此,首先将SecYEG和/或YidC重构为不含DAG的蛋白脂质体,然后通过冻融-超声处理将其与含有DAG和MPIase的脂质体进行膜融合,从而产生单层蛋白脂质体(Sasaki 等,2019)。这样的蛋白脂质体准备用于膜整合测定。该测定系统包括Pure System,一种重组翻译系统,用于体外合成底物膜蛋白(Nishiyama 等,2010;Shimizu 等,2001)。通过分析膜保护片段(MPF)来确定整合活性(Koch 等,1999)。整合反应后蛋白酶消化产生的MPF反映了膜的整合。

关键字:膜蛋白整合, SecYEG, YidC, 膜蛋白整合酶, 信号识别颗粒, 甘油二脂, 蛋白质脂质体

材料和试剂


 


1. 塑料管(1.5毫升)(润滑脂,目录号:616201)      


2. 超速离心管(1.5毫升)(Beckman Coulter,目录号:357448)      


3. 玻璃小瓶(2毫升)(一次购,目录号:9-852-01)      


4. 透析管(Tokyo Garasu Kikai Co.,Ltd,目录号:03272337)      


5. 大肠杆菌极性脂质提取物(磷脂)(Avanti Polar Lipids,Inc.,目录号:100600C)      


6. 1,2-二油酰基-sn- 甘油(DAG)(默克,目录号:D0138)      


7. 2- [4-(2-羟乙基)-1-哌嗪基] 乙磺酸(HEPES)(Dojindo Laboratories,目录号:342-01375)      


8. 二硫苏糖醇(Wako,目录号:048-29224)      


9. 氯仿,保证试剂(和光,目录号:038-02606)      


10. 丙酮,保证试剂(和光,目录号:012-00343)   


11. Ñ - 辛基- β - D- 吡喃葡萄糖苷(OG)(同仁化学实验室,目录号:O001)   


12. [ 35 S] -EXPRESS蛋白标记混合物(PerkinElmer Inc,目录号:NEG072)   


13. 纯系统(基因前沿,目录号:PF001-0.25)   


14. 蛋白酶K(罗氏(Roche),目录号:03115879001)   


15. 三氯乙酸,保证试剂(和光,目录号:204-02405)   


16. 丙烯酰胺,电泳级(和光,目录号:011-08015)   


17. Ñ ,Ñ “ - 亚甲基双(丙烯酰胺),电泳级(和光,目录号:130-06031)   


18. 十二烷基硫酸钠(SDS),用于分子生物学(Wako,目录号:194-13985)   


19. 2-氨基-2-羟甲基-1,3-丙二醇(Tris),保证试剂(和光,目录号:207-06275)   


20. Na 2 HPO 4 ,保证试剂(和光,目录号:197-02865)   


21. NaH 2 PO 4 ,保证试剂(和光,目录号:192-02815)   


22. 甘氨酸,保证试剂(和光,目录号:077-00735)   


23. 溴酚蓝(和光,目录号:021-02911)   


24. β - 巯基乙醇(和光,目录号:131-14572)   


25. 甘油,保证试剂(和光,目录号:075-00611 )   


26. Ffh,如所述纯化(Eisner 等,2003)   


27. FtsY,如所述纯化(Koch 等,1999)   


28. YidC,如所述纯化(Nishikawa 等,2017)   


29. SecYEG,如所述纯化(Moser 等,2013)   


30. MPIase,如所述纯化(Nishiyama 等,2010和2012 )   


31. TALON金属亲和树脂(Clontech ,目录号:635503)   


32. 成像板(GE,BAS IP MS 2025 E,目录号:28-9564-75)   


33. 缓冲区A(请参阅食谱)   


34. OG库存(请参阅食谱)   


35. 磷酸钠缓冲液(pH 7.2)(请参见食谱)   


设备


 


浴式超声波仪(布兰森,型号:Bransonic 12 )
冷冻干燥机(EYELA,型号:FD-80)
真空泵(ULVAC,型号:GCD-051X)
超速离心机(贝克曼库尔特公司,型号:Optima TL)
转子(贝克曼库尔特,型号:TLA-55)
保温箱(Eppendorf,型号:Thermomixer Comfort)
Phosphorimager(GE,型号:Storm 820)
-80 °C冷冻室
 


软件


 


ImageQuant软件(GE Healthcare)
 


程序


 


含有蛋白质因子如SecYEG和YidC的蛋白脂质体的制备
将磷脂溶液(25 mg / ml 的氯仿溶液)放在玻璃瓶中。对于一个重建样本,请使用200 μ克磷脂。在N 2 气流下向上干燥磷脂,直到大部分氯仿蒸发为止,然后在真空下的冷冻干燥器中放置1小时。
暂停在缓冲液A(50mM的HEPES-KOH,pH7.5的,1mM的磷脂(200微克/一个重构样品)二硫苏糖醇在)10毫克/米升,接着浴超声处理(约10秒),以形成脂质体,直到得到稍微不透明的悬浮液。
通过添加15%[w / v]的储备液,在1.5 %[w / v] OG的缓冲液A中增溶脂质体。
混合溶解磷脂(200微克)与SecYEG (〜10 μ 克)和/或YidC (〜10 μ 克在1.5%溶解)[W / V] OG。将体积调节至100〜μ 升,保持OG浓度为1.5%[w / v的] 。将混合物在冰上孵育20分钟。
如果将SecYEG和YidC溶解在DDM或DPC中,则将其用于溶解SecYEG和YidC的清洁剂更改为OG。将样品上样至TALON柱,然后用含1.5%[w / v] OG的缓冲液洗涤,然后用含100 mM咪唑的缓冲液洗脱。对于SecYEG,将40%[w / v] 甘油添加到缓冲液中。保持DDM / DPC浓度尽可能低。
在4°C下用500 ml 缓冲液A 透析混合物至少3 h,以除去OG 形成蛋白脂质体。蛋白脂质体悬浮液变得有点混浊。
向透析的样品中加入0.9 ml 的缓冲液A,然后通过离心(170,000 x g ,1 h,4 °C)回收蛋白脂质体。重悬粒料50μ 升B的uffer甲。
通过冷冻(在液氮中)-融化(在室温下)-超声处理(约10 s)一次,使脂质体成为单层。超声处理后,请确保悬浮液未加热。
解冻和超声处理之前,如有必要,将冷冻的脂质体保存在-80 °C。
 


含DAG的脂质体的制备。
混合磷脂(1 mg)和DAG(0.05 mg),并按照步骤A1干燥脂质混合物。
暂停在缓冲液A将干燥的残余物以1个0毫克磷脂/米升,接着浴超声处理(〜10秒)。
 


含有DAG和MPIase的脂质体的制备
按照步骤B1所述,将磷脂和DAG 在氯仿中混合,然后将混合物干燥。通常,将25%[w / w]的DAG添加到磷脂中。
将混合物溶解在溶剂C中(氯仿/乙醇/水= 3/7/4),然后与溶解在溶剂C中的MPIase混合,通常相对于磷脂为25%[w / w]。
在N 2 流下蒸发溶剂C ,然后在真空下蒸发。
悬浮在干燥的残余物的缓冲液A在1个0毫克磷脂/米升,接着浴超声处理(〜10秒)。
 


蛋白脂质体与MPIase / DAG-脂质体融合以掺入MPIase和DAG
将步骤A中获得的蛋白脂质体悬浮液与MPIase / DAG-脂质体(步骤C)混合,通常以4:1的比例混合,可得到含有5%[w / w] DAG和5%[w / w] MPIase的蛋白脂质体。
重复冷冻(在液氮中)-解冻(在室温下)-超声处理(约10 s)的循环三次,以使脂质体融合。通过这种方式,可以制备含有SecYEG,YidC和MPIase / DAG的蛋白脂质体。 
 


(3L-)Pf3涂层整合的反应(图1)
翻译体外底物蛋白,PF3涂层和亩TANT 3L-PF3涂层(基弗和库恩,1999 ; Serek 。等人,2004),使用纯系统,重构无细胞系统在体外翻译(西山等等人,2010;Shimizu 等人,2001)。Pure System(20 µl )的反应混合物包含[ 35 S]蛋氨酸(2.5〜10 MBq / ml ;〜200 nM),质粒DNA(pT7-3L-Pf3或pT7-Pf3 [ Kawashima 等, 2008;Serek 等,2004 ] ;〜20 µg / ml )和(蛋白)脂质体(0.4 mg磷脂/ ml )(表1)。当需要高水平的合成时,向混合物中加入冷蛋氨酸(0.3 mM)。在这些条件下,观察到合成水平提高了约200倍。PURE系统是可商购自基因前沿,但调整缓冲系统customly 如所描述的(西山等人,2010)。为避免蛋白脂质体聚集,请确保Mg浓度为9 mM或更低,并且反应混合物中不包含PEG和多胺,例如腐胺和亚精胺。
在37°C下进行30分钟的翻译/整合反应。
通过在冰上冷却5分钟终止反应。将3 µl 和15 µl 的混合物(20 µl )倒入新管中。
将3 µl 三氯乙酸(10%[w / v] )加入等分试样(3 µl )中以沉淀合成的蛋白质。
将等体积的蛋白酶K(1 mg / ml )与另一等分试样(15 µl )混合,然后在25 °C 孵育25分钟以生成MPF。
蛋白酶K消化后,添加30 µl 10%[w / v] 三氯乙酸,然后在56°C孵育5分钟以灭活蛋白酶K。在冰上孵育至少10分钟以沉淀MPF。
通过离心(10,000 xg ,持续5分钟)回收沉淀物(St eps E 4和E 6 ),然后用100%丙酮洗涤。离心(10,000 xg ,5分钟)后,将沉淀在室温下干燥20〜30 分钟。
将沉淀物溶于10 µl SDS样品上样缓冲液A中,然后煮沸3分钟。SDS样品上样缓冲液A由25 mM磷酸钠(pH 7.2),2.5%[w / v] SDS,25%[w / v] 甘油,2.5%[w / v] β- 巯基乙醇和0.0125%组成[w / v] 溴酚蓝。
如Nishiyama 等人 所述,将样品加到含有6 M尿素的SDS凝胶上。(2006)。凝胶是由12.5%[W / V] 丙烯酰胺0.27%[W / V] Ñ ,Ñ “ - 亚甲基双(丙烯酰胺)在100 毫如描述磷酸钠缓冲液(pH 7.2)侯赛因等。(1980)。  
电泳后,将凝胶浸入10%[v / v] 乙酸中10分钟,然后干燥。
将凝胶暴露在暗盒中的成像板上过夜。
 


表1.纯系统的组成


能源


 


2毫米


ATP,GTP


1毫米


CTP,UTP


20毫米


磷酸肌酸


缓冲液


 


50毫米


HEPES - KOH,pH 7.6


100毫米


谷氨酸钾


9毫米


醋酸镁


1毫米


DTT


其他组件


 


0.3毫米


除蛋氨酸外有19个氨基酸


10毫克/毫升


10-甲酰基-5,6,7,8-四氢叶酸


56 A 260 nm /毫升


混合RNA


 


酶混合物(1/20稀释)


1微米


核糖体


〜20微克/立方米升


质粒DNA


约200 nM


[ 35 S]蛋氨酸


0.4毫克/米升


(蛋白)脂质体


 


D:\ Reformatting \ 2020-3-2 \ 1396--1903033西山健一837295 \ Figs jpg \ Fig1.jpg


图1.(3L- )Pf3 涂层整合的测定方案


 


MtlA(甘露醇渗透酶)整合反应
使用pET-MtlA (Kuruma et al。,2005)和Pure System 合成E中的MtlA 。向反应混合物中加入Ffh(50 µg / ml ),FtsY(17 µg / ml )和冷蛋氨酸(30 µM)。
在37°C下进行翻译/整合反应30分钟后,将5%[w / v] 三氯乙酸加入到等分试样(9 µl )中,并按照步骤3中所述用蛋白酶K 消化另一等分试样(9 µl )。小号E4- è 如步骤E5描述5.恢复沉淀。
溶解干燥的沉淀物在10 μ 升SDS样品上样缓冲液B的,随后煮沸3分钟。SDS样品上样缓冲液B由62.5 mM Tris-HCl(pH 6.8),3%[w / v] SDS,10%[w / v] 甘油,5.5%[w / v] β- 巯基乙醇和0.01组成%[w / v] 溴酚蓝。
如所述(Laemmli,1970)将样品加载到SDS-凝胶上。凝胶是由15%[w / v的] 丙烯酰胺的0.4%[w / v的] Ñ ,Ñ ' - 亚甲基双(丙烯酰胺)。
电泳后,将凝胶浸入10%[v / v] 乙酸中10分钟,然后干燥。将凝胶放在成像板上。
 


数据分析


 


确定整合活动


使用Phosphorimager检测合成底物和MPF的放射性谱带。
使用ImageQuant软件确定每个波段的放射性(图2)。使用软件手动减去背景水平。
通过将MPF水平除以合成底物的MPF水平来确定整合活性(图2,每个凝胶图像的底部)。考虑底物和MPF中蛋氨酸的数量。
 


D:\ Reformatting \ 2020-3-2 \ 1396--1903033西山健一837295 \ Figs jpg \ Fig 2.jpg


图2.完整定量分析结果的示例。一。Pf3涂层整合取决于MPIase,并受YidC刺激。乙。MPIase和SecYEG都是MtlA集成必不可少的。此外,YidC刺激MtlA的MPIase / SecYEG依赖性整合。蛋氨酸的数目(MtlA为25,MtlA-MPF为18)被用于活性测定。图像取自Sasaki 等人(2019年)(图3C和3D)。


 


菜谱


 


缓冲液A
用ddH 2 O将1 M HEPES-KOH(pH 7.2)和1 M二硫苏糖醇溶液分别稀释至50 mM和1 mM


OG库存
以15%[w / v]将OG溶解在ddH 2 O中,并在4 °C下储存


所以dium磷酸盐缓冲液(pH 7.2)中
准备1 M NaH 2 PO 4 和1 M Na 2 HPO 4 溶液
混合这些溶液,监测pH值,得到1 M 磷酸钠缓冲液(pH 7.2)
室温下的储备溶液
 


Acknowledg 发言:


 


这项工作得到了日本促进科学技术援助补助金协会18J21847(对HN)的支持;15KT0073、16H01374、16K15083、17H02209和18KK0197(针对KN)。该方案源自我们的报告(Sasaki et al。,2019)。


 


利益争夺


 


我们声明我们没有利益冲突。


 


参考文献


 


Alami,M.,Trescher,D.,Wu,LF和Muller,M.(2002)。大肠杆菌中双精氨酸易位(Tat)特异性膜结合和易位的单独分析。生物化学杂志277(23):20499-20503。
I.Collinson,Breyton,C.Duong,F.Tziatzios,C.Schubert,D.Or,E.,Rapoport,T。和Kuhlbrandt,W。(2001)。细菌核心蛋白转位酶的投影结构和寡聚特性。Embo J 20(10):2462-2471。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Nishikawa, H., Sasaki, M. and Nishiyama, K. (2020). In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes. Bio-protocol 10(10): e3626. DOI: 10.21769/BioProtoc.3626.
  2. Sasaki, M., Nishikawa, H., Suzuki, S., Moser, M., Huber, M., Sawasato, K., Matsubayashi, H. T., Kumazaki, K., Tsukazaki, T., Kuruma, Y., Nureki, O., Ueda, T. and Nishiyama, K. I. (2019). The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins. J Biol Chem 294(49): 18898-18908. 
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