实验方法来研究线粒体定位和核细胞周期激酶CDK1功能
Summary
Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.
Abstract
Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2′-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.
Introduction
在哺乳动物中，细胞周期进展取决于由细胞周期蛋白和周期蛋白依赖性激酶（的Cdks）1控制的高度有序的事件。通过其胞质，细胞核，和中心体定位，其CyclinB1 / CDK1能够在有丝分裂的不同的事件，如核膜破裂和中心体分离2同步。其CyclinB1 / CDK1保护有丝分裂细胞抗凋亡3，促进线粒体分裂，线粒体平均分配给新成立的子细胞4的关键一步。
在增殖哺乳动物细胞中，线粒体ATP经由氧化磷酸化（OXPHOS）机械（电子传递链），它是由5多亚基复合物生成;复杂的I – 复杂V（CI-CV）。烟酰胺腺嘌呤二核苷酸（NADH）：泛醌氧化还原酶或复合物I（CI）是理解的五个配合物5的最大和最小。在复杂的C的45亚基，其中14 onsists形成催化核心。一旦组装好，复杂的假定的L形结构有一个臂突出到基体和嵌入在内膜6,7-另一个臂。在CI亚基突变是多种线粒体紊乱8的原 因。在OXPHOS一支高效CI不仅需要整体线粒体呼吸9，而且对成功的细胞周期进程10。 Unravelling在健康和疾病这种膜结合酶复合物的功能所依据的机制可以启用新的诊断程序和先进的治疗策略的开发。在最近的研究中，我们已经发现，中CyclinB1 / CDK1复杂易位到在（峡2），G2 /（有丝分裂）M期线粒体和磷酸CI亚基以提高线粒体的能量生产，潜在地抵消细胞中的细胞的增加的能源需求周期11。在这里，我们笑wcase实验程序，并且可以被用来研究否则核/质激酶的线粒体易位策略，其线粒体底物，以及使用其CyclinB1 / CDK1，例如他们的线粒体定位的功能的后果。
这一发现在需要的时候提示特定线粒体表达这种复杂的击倒的研究中CyclinB1 / CDK1复杂易位到线粒体。以实现蛋白质的特异性线粒体表达，人们可以在感兴趣的蛋白的N-末端添加一个线粒体靶向序列（MTS）。线粒体靶向序列使线粒体蛋白到他们正常居住12线粒体的排序。我们使用来自人的细胞色素C氧化酶亚基8A（COX8）的前体衍生的87碱基线粒体靶向序列，并克隆到绿色荧光蛋白（GFP）-tagged CyclinB1的或红色荧光蛋白（RFP）-tagged含质粒的帧CDK1。该方法使我们能够针对CyclinB1的和CDK1进入线粒体，具体地改变这些蛋白质的线粒体表达而不影响它们的核池。通过荧光标记这些蛋白质，我们能够监视他们的实时定位。同样，我们推出了MTS到含有RFP标记的显性负CDK1的质粒，使我们能够明确打掉CDK1的线粒体表达和功能。它具有双本地化一样CDK1激酶的线粒体和核函数来区分是必不可少的。工程MTS到这些双功能激酶的N端提供了一个很好的策略，很容易被采用和有效的。
因为CDK1是细胞周期激酶，它是基本的，以确定细胞周期进展时CDK1被本地化为线粒体。为了实现这一目标，我们已利用的新方法具d，来监测细胞中的DNA含量。传统的方法包括使用的BrdU（溴脱氧尿苷），一种合成的胸苷类似物，它在细胞周期的S期合并到新合成的DNA代替胸苷。然后正在积极复制它们的DNA的细胞可以使用抗BrdU抗体进行检测。这种方法的一个缺点是，它需要的DNA变性通过苛刻的方法，如酸或热处理，这可能导致在结果中13,14之间的不一致性，以提供所述的BrdU抗体的访问。另外，我们采用了类似的方法，以监测不同的胸苷类似物，埃杜的积极分裂的细胞。检测的EdU不需要苛刻的DNA变性，中性清洁剂处理使检测试剂来访问这些EDU在新合成的DNA。所述的EdU方法已被证明是更可靠的，一致的，并与高通量分析15的潜力。
最后，T○确定CDK1的线粒体基质中，我们使用了一种名为2D-DIGE蛋白质组学的工具，这是经典的双向电泳的高级版本。二维电泳根据在第二第一维和分子量等电点分离蛋白。由于翻译后修饰，如磷酸化影响的蛋白质的等电点和分子量，2D凝胶可检测不同样品中的蛋白质的磷酸化状态之间的差异。蛋白质的大小（面积和强度）掩护变化与蛋白质的表达水平，允许多个样品间的定量比较。使用这种方法，我们能够区分磷酸化蛋白质在野生型与突变线粒体靶向CDK1表达细胞。特定蛋白质斑点，在野生型显示，但在线粒体靶向突变CDK1制备失踪分离并通过质谱法鉴定。
在传统的2D凝胶，三苯甲烷染料被用来可视化凝胶上的蛋白质。 2D-DIGE使用荧光蛋白标签与蛋白质的电泳迁移率的影响最小。不同的蛋白质样品可以用不同的荧光染料，混合在一起，并通过相同的凝胶上分离，从而允许在单一凝胶16多个样品的共电泳进行标记。这最大限度地减少了凝胶到凝胶变体，它是在基于凝胶的蛋白质组学研究的一个关键问题。
Protocol
Representative Results
Discussion
往其他亚细胞蛋白质一样，线粒体有针对性的蛋白质具有自己的原发性或继发性结构中的定位信号，即引导他们以精心的蛋白质易位和折叠机21,22的帮助下，细胞器。线粒体靶向从独占线粒体驻地蛋白质如COX8获得的序列（MTS）可以被添加到任何基因序列的N末端 至特异性蛋白靶向进入线粒体11,23,24。这里，其CyclinB1和CDK1基因克隆到含有载体COX8 MTS和表达时，重组CyclinB1的和CDK1被…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH grants CA133402, CA152313 and Department of Energy Office of Science DE-SC0001271. We thank the University of California Davis Flow Cytometry Shared Resource Laboratory with funding from the NCI P30 CA0933730, and NIH NCRR C06-RR12088, S10 RR12964 and S10 RR 026825 grants and with technical assistance from Ms. Bridget McLaughlin and Mr. Jonathan Van Dyke for their help with the flow cytometry experiments.
Materials
| 32P ATP | PerkinElmer | BLU002001MC | |
| Anti-mouse secondary antibody | Invitrogen | A-11003 | Alexa-546 conjugated |
| Anti-rabbit secondary antibody | Invitrogen | A11029 | Alexa-488 conjugated |
| ATP | Research Organics | 1166A | For in vitro kinase assay |
| Cdk1 antibody | Cell Signaling Technology | 9112 | |
| Cdk1 kinase buffer | New England Biolabs | P6020S | |
| Click-iT EdU Alexa Fluor 488 Imaging Kit | Life Technologies | C10337 | For cell cycle analysis with EdU labeling |
| COX IV antibody | Cell Signaling Technology | 4844S | For mitochondrial immunostaining |
| Cyclin B1 antibody | Santa Cruz Biotech | sc-752 | |
| CyclinB1/Cdk1 enzyme complex | New England Biolabs | P6020S | Avoid freeze/thaw |
| CyDye DIGE Fluor Labeling Kit | GE Healthcare Life Sciences | 25-8009-83 | |
| DIGE Gel and DIGE Buffer Kit | GE Healthcare Life Sciences | 28-9480-26 AA | |
| Dimethylformamide | Sigma Aldrich | 319937 | DMF |
| Dithiothreitol | Bio-Rad | 161-0611 | DTT |
| dNTP | EMD Millipore | 71004 | For site-directed mutagenesis |
| Dpn I enzyme | Stratagene | 200519-53 | For site-directed mutagenesis |
| Dry Strip cover fluid | GE Healthcare Life Sciences | 17-1335-01 | Used as mineral oil |
| EDTA | J.T. Baker | 4040-03 | |
| EGTA | Acros Organics | 409910250 | |
| Eppendorf Vacufuge Concentrator | Fisher Scientific | 07-748-13 | Used as vacuum centrifuge concentrator |
| Fluoromount G | Southern Biotech | 0100-01 | Anti-fade mounting solution |
| Fortessa Flow Cytometer | BD Biosciences | 649908 | For cell cycle analysis with EdU labeling |
| Histone H1 | Calbiochem | 382150 | For in vitro kinase assay |
| QIAquick Gel Extraction Kit | Qiagen | 28704 | For purifying DNA fragments from agarose gels |
| Immobiline DryStrip Gels | GE Healthcare Life Sciences | 18-1016-61 | IEF (isoelectric focusing) strips |
| Immobilized Glutathione | Thermo Scientific | 15160 | Glutathione-agarose beads |
| Iodoacetamide | Sigma Aldrich | I1149 | IAA |
| IPGphor 3 Isoelectric Focusing Unit | GE Healthcare Life Sciences | 11-0033-64 | IPGphor strip holders |
| Isopropyl-b-D-thio-galactopyranoside | RPI Corp | 156000-5.0 | IPTG |
| Leupeptin | Sigma Aldrich | L9783 | For cell lysis buffer |
| Lipofectamine 2000 | Life Technologies | 11668027 | Transfection reagent |
| Lysine | Sigma Aldrich | L5501 | For CyDye labeling |
| Lysozyme | EMD Chemicals | 5960 | |
| Mitoctracker Red/Green | Invitrogen | M7512/M7514 | Mitochondrial fluorescent dyes |
| MOPS | EMD Chemicals | 6310 | |
| pEGFP-N1 | Clonetech | 6085-1 | GFP-expressing vector |
| Pfu | Stratagene | 600-255-52 | |
| pGEX-5X-1 | GE Healthcare Life Sciences | 28-9545-53 | GST-expressing vector |
| Phenylmethylsulfonyl fluoride | Shelton Scientific | IB01090 | PMSF |
| Phosphate buffered saline | Life Technologies | 14040 | PBS |
| Spectra/Por 4 dialysis tubing | Spectrum Labs | 132700 | as porous membrane tubing for dialysis |
| Pro-Q Diamond Phosphoprotein Gel Stain | Life Technologies | P-33300 | For staining phosphoproteins on 2D gels |
| Proteinase inhibitor cocktail | Calbiochem | 539134 | For cell lysis buffer |
| QuikChange site-directed mutagenesis kit | Stratagene | 200519-5 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27104 | MiniPrep Plasmid Isolation Kit |
| RO-3306 | Alexis Biochemicals | 270-463-M001 | Cdk1 inhibitor |
| Rotenone | MP Biomedicals | 150154 | Complex I inhibitor |
| Sodium carbonate | Fisher Scientific | S93359 | |
| Sodium chloride | EMD Chemicals | SX0420-5 | For cell lysis buffer |
| Sodium orthovanadate | MP Biomedicals | 159664 | For cell lysis buffer |
| Sodium pyrophosphate decahydrate | Alfa Aesar | 33385 | For cell lysis buffer |
| Sodium β-glycerophosphate | Alfa Aesar | L03425 | For cell lysis buffer |
| SpectraMax M2e | Molecular Devices | M2E | Microplate reader |
| Sucrose | Fisher Scientific | 57-50-1 | |
| Tissue Grinder pestle | Kimble Chase | 885301-0007 | For mitochondria isolation |
| Tissue Grinder tube | Kimble Chase | 885303-0007 | For mitochondria isolation |
| Trichloroacetic acid solution | Sigma Aldrich | T0699 | TCA |
| Tris | MP Biomedicals | 103133 | |
| Triton-x-100 | Teknova | T1105 | |
| Trypsin | Calbiochem | 650211 | |
| Typhoon Imager | GE Healthcare Life Sciences | 28-9558-09 | Laser gel scanner fro 2D-DIGE |
| Ubiquinone | Sigma Aldrich | C7956 |
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