The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and axe similar to acidocalcisomes
The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and axe similar to acidocalcisomes
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DOI：
10.1074/jbc.m105268200
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发表时间：
2001-12-07
影响因子：
4.8
通讯作者：
Docampo, R
中科院分区：
文献类型：
--
作者：
Ruiz, FA;Marchesini, N;Docampo, R
酸钙体是最初在锥虫和顶复门寄生虫中描述的酸性钙储存室。在这项工作中，我们描述了细胞器的性质类似于酸钙体在绿色衣藻莱茵衣藻。白霉素和NH 4Cl从预先装载的透化细胞中释放Ca-45（2+），表明大量的这种阳离子掺入酸性隔室中。用X射线微区分析法对C.莱因哈氏藻显示出大量的磷、镁、钙和锌。免疫荧光显微镜，使用抗拟南芥液泡型质子焦磷酸酶（H+-PPase）肽序列的抗血清，该肽序列在C. reinhardtii酶，表明定位于质膜、细胞内液泡和收缩液泡中，在收缩液泡中与液泡质子ATP酶（V-H+-ATP酶）共定位。使用碘克沙醇密度梯度纯化的电子致密液泡表明，除了高浓度的PPi和短链和长链多磷酸盐外，H+-PPase和V-H+-ATPase活性优先定位，但缺乏线粒体和叶绿体的标记物。在孤立的电子致密的液泡中，PPi驱动的质子转运被钾离子刺激，并被PPi类似物aminomethylenediphosphonate抑制。氟化钾、亚氨二磷酸、N，N '-二环己基碳二亚胺和N-乙基马来酰亚胺也以剂量依赖性方式抑制分离的细胞器中PPi的水解。这些结果表明，C。reinhardtii在化学组成和质子泵的存在方面与酸钙体非常相似。多磷酸也定位于收缩泡的4 '，6-diamidino-2-phenylindole染色，这表明，与免疫化学数据，这些细胞器和酸钙体之间的联系。
Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH4Cl released Ca-45(2+) from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H+-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H+-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H+-PPase and the V-H+-ATPase activities in addition to high concentrations of PPi and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PPi-driven proton translocation was stimulated by potassium ions and inhibited by the PPi analog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PPi hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4',6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.