In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves
对苦草属叶片微粒体和浆膜切片中的潜在蓝光受体向光素进行体外磷酸化测定
发布: 2015年11月05日第5卷第21期 DOI: 10.21769/BioProtoc.1647 浏览次数: 9584
评审: Samik BhattacharyaSam-Geun KongAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2015
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Abstract
An aquatic angiosperm Vallisneria (Alismatales: Hydrocharitaceae) has been used as an excellent experimental material over a century to study the light regulation of dynamic intracellular movements including chloroplast redistribution and cytoplasmic streaming (Senn, 1908; Seitz, 1987; Takagi, 1997). However, understanding of the molecular mechanisms lagged behind because of difficulty in applying modern techniques such as gene transformation to this plant. Especially, which kind of photoreceptors function in these intriguing responses has long been an unsolved topic. Recently, genes encoding plant-specific blue-light receptor phototropins were isolated in Vallisneria, for the first time from aquatic plants (Sakai et al., 2015). Phototropins were identified first as the photoreceptor for hypocotyl phototropism in Arabidopsis thaliana, and now known to regulate many responses including chloroplast photorelocation movements in various plant species (Christie, 2007). Phototropins are localized mainly on the plasma membrane and their auto-phosphorylation induced by blue light is the critical step of signal transduction pathway (Sakamoto and Briggs, 2002; Kong et al., 2006; Kong et al., 2013; Inoue et al., 2010). Here we describe a protocol for in vitro protein phosphorylation assay using crude-microsomal and plasma-membrane-enriched fractions of Vallisneria, which enabled us to verify the presence of phototropins and characterize their auto-phosphorylation responses. After these analyses, Sakai et al. (2015) proposed that Vallisneria phototropins mediate the high-intensity-blue-light-induced chloroplast avoidance response.
Keywords: Crude microsomal fraction (粗微粒体级分)
Plasma-membrane-enriched fraction (血浆膜富集级分)
Light-induced phosphorylation (光诱导的磷酸化)
Aquatic plants (水生植物)
SDS-PAGE (SDS-PAGE)
Materials and Reagents
- Commonly-used medical cotton gauze
- 0.2 ml tube
- Vallisneria plants
Note: Young plants of Vallisneria of 20-30 cm long were purchased at a tropical-fish store and cultured to be grown over 100 cm in buckets (20 L) filled with tap water and with a layer of soil at the bottom (Figure 1A). The plants were grown under 12 h light/12 h dark regime at 20-24 °C. - Dextran (from Leuconostoc ssp., Mr ~500,000) (Fluka, catalog number: 31392 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 31392 ”. - Polyethylene glycol (Sigma-Aldrich, catalog number: P-3640 )
- SDS-PAGE
- Deionized water
- 1% Triton X-100 (Sigma-Aldrich, catalog number: T-9284 )
- 50 mM MOPS-KOH (pH7.6) (DOJINDO, catalog number: 343-01805 )
- 300 mM sucrose (Wako Pure Chemical Industries, Siyaku, catalog number: 196-00015 )
- 10 mM ethylene glycol bis (2-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid (EGTA) (DOJINDO, catalog number: 342-01314 )
- 5 mM ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865 )
- 10 μg/ml dibutylhydroxytoluene (Tokyo Chemical Industry, catalog number: D0228 )
- 1% casein (Nacalai tesque, catalog number: 073-19 )
- 5 mM K2S2O5 (Wako Pure Chemical Industries, Siyaku, catalog number: 161-03345 )
- 1 mM dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 048-29224 )
- 2.5 mg/ml pepstatin A (Sigma-Aldrich, catalog number: P-4265 )
- 2.5 mg/ml aprotinin (Sigma-Aldrich, catalog number: A-4529 )
- 20 mg/ml EDTA-washed Polyvinylpolypyrolidone (Sigma-Aldrich, catalog number: P-6755 )
- MOPS-KOH (DOJINDO, catalog number: 345-02225 )
- 25 mM MgCl2 (Wako Pure Chemical Industries, Siyaku, catalog number: 135-00165 )
- 250 mM KCl (Wako Pure Chemical Industries, Siyaku, catalog number: 163-03545 )
- 40% glycerol (Wako Pure Chemical Industries, Siyaku, catalog number: 075-00616 )
- 20% 2-mercaptoethanol (Sigma-Aldrich, catalog number: A-6365 )
- A small amount of bromophenol blue (Wako Pure Chemical Industries, Siyaku, catalog number: 021-02911 )
- 0.5 M Tris-HCl (pH 6.8) (Sigma-Aldrich, catalog number: 252859 )
- Homogenizing medium (see Recipes)
- Buffer A (see Recipes)
- Buffer B (see Recipes)
- 5x phosphorylation buffer (see Recipes)
- ATP mixture (see Recipes)
- 4x SDS buffer (see Recipes)
Equipment
- Blade (Lion Office Products Corp., model: L-300 )
- Polytron homogenizer (Kinematica AG, model: PT-35/ 2ST”OD” )
- Centrifuge
- Ultracentrifuge
- Teflon homogenizer (10 ml) (Ikemoto Scientific Technology, model: 812-771-04 )
- Lighting system (Sugiura Lab, model: FI-150T )
- Cut-off filter (KenkoTokina Corporation, model: Y-44 )
- Interference filter (KenkoTokina Corporation, model: BP-45 )
- Autoradiography (Fujifilm Corporation)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Sakai, Y., Inoue, S. and Takagi, S. (2015). In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves. Bio-protocol 5(21): e1647. DOI: 10.21769/BioProtoc.1647.
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分类
植物科学 > 植物细胞生物学 > 细胞器分离
生物化学 > 蛋白质 > 修饰
细胞生物学 > 细胞信号传导 > 磷酸化
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