QUANTIFICATION AND LOCALIZATION OF PHOSPHORYLATED MYOSIN-I ISOFORMS IN ACANTHAMOEBA-CASTELLANII
QUANTIFICATION AND LOCALIZATION OF PHOSPHORYLATED MYOSIN-I ISOFORMS IN ACANTHAMOEBA-CASTELLANII
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DOI：
10.1083/jcb.130.3.591
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发表时间：
1995-08-01
影响因子：
7.8
通讯作者：
KORN, ED
中科院分区：
文献类型：
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作者：
BAINES, IC;CORIGLIANOMURPHY, A;KORN, ED
肌动蛋白激活的Mg~（2+）-ATP酶活性的三个肌球蛋白I亚型在卡氏阿米巴中仅在肌球蛋白I重链的单个位点磷酸化后显著表达。合成的磷酸化和未磷酸化的肽对应的磷酸化位点序列，不同的三个肌球蛋白I亚型，被用来提高亚型特异性抗体，仅识别磷酸化的肌球蛋白I或总肌球蛋白I亚型（磷酸化和未磷酸化），分别。用这些抗血清，总的和磷酸化的亚型的数量进行了定量，磷酸肌球蛋白I亚型的本地化，和房室分布的磷酸肌球蛋白亚型determined.Myosin IA，这是几乎完全在肌动蛋白丰富的皮质，是70 - 100%的磷酸化，特别是在吞噬杯富集。肌球蛋白IB和IC主要与质膜和大空泡膜相关，在那里它们仅10 - 20%磷酸化，而细胞质肌球蛋白IB和IC与细胞质肌球蛋白IA一样，大部分磷酸化（60 - 100%）。此外，磷酸肌球蛋白IB集中在质膜的活动区域。与收缩空泡（CV）膜相关的磷酸肌球蛋白IC和F-肌动蛋白比填充CV膜多20倍以上。由于CV相关肌球蛋白IC的总量保持恒定，因此它必须在CV收缩开始时被磷酸化。这些数据扩展了先前关于阿米巴中肌球蛋白I同工酶的特定功能的建议（Baines，I. C.的方法，H. Brzeska和E. D.克恩。1992. 119：1193 - 1203）：吞噬作用中的磷酸肌球蛋白IA，吞噬作用和胞饮作用中的磷酸肌球蛋白IB，以及CV收缩中的磷酸肌球蛋白IC。
The actin-activated Mg2+-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined.Myosin IA, which was almost entirely in the actin-rich cortex, was 70-100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-foId more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I. C., H. Brzeska, and E. D. Kern. 1992. J. Cell Biol. 119:1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.