Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis
Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis
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DOI：
10.1104/pp.103.020008
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发表时间：
2003-08-01
期刊：
影响因子：
7.4
通讯作者：
Harper, JF
中科院分区：
文献类型：
--
作者：
Dammann, C;Ichida, A;Harper, JF
钙依赖性蛋白激酶（CDPKs）是植物和某些原生生物所特有的。它们被钙激活使其成为细胞内钙信号转导的重要开关。在这里，我们确定了9个CDPK异构体的亚细胞靶向潜力，从拟南芥，通过在转基因植物中表达的绿色荧光蛋白（GFP）融合。通过荧光显微镜在根尖附近的细胞中确定亚细胞位置。同种型AtCPK 3-GFP和AtCPK 4-GFP显示出与游离GFP相似的核和胞质分布。膜分离实验证实，这些亚型主要是可溶性的。基于成像和膜分离实验，观察到AtCPKs 1、7、9、9、16、21和28的膜缔合。这与潜在的N-末端酰化位点的存在相关，与酰化作为膜结合中的重要因素一致。除了一种以外，所有的膜相关亚型都专门靶向质膜，例外是AtCPK 1-GFP，其靶向过氧化物酶体，如通过与过氧化物酶体标记物的共可视化所确定的。AtCPK 1-GFP的过氧化物酶体靶向被两个潜在的N-末端酰化位点的缺失破坏。过氧化物酶体定位的CDPK的观察表明钙调节过氧化物酶体功能的机制涉及氧化应激和脂质代谢。
Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 9, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane, The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.