Interactions between the yeast SM22 homologue Scp1 and actin demonstrate the importance of actin bundling in endocytosis.
Interactions between the yeast SM22 homologue Scp1 and actin demonstrate the importance of actin bundling in endocytosis.
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DOI：
10.1074/jbc.m710332200
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发表时间：
2008-05-30
影响因子：
4.8
通讯作者：
Ayscough, Kathryn R.
中科院分区：
文献类型：
--
作者：
Gheorghe, Dana M.;Aghamohammadzadeh, Soheil;Rooij, Lwona I. Smaczynska-de;Allwood, Ellen G.;Winder, Steve J.;Ayscough, Kathryn R.
酵母SM22同源物Scp1先前已被证明在体外作为肌动蛋白捆绑蛋白。在细胞中，Scp1定位于作为内吞过程中内翻过程的一部分形成的皮质肌动蛋白斑块，其功能与已被充分表征的酵母纤维蛋白同源物Sac6p重叠。在这项工作中，我们使用活细胞成像来证明Scp1肌动蛋白界面中关键残基的重要性。我们在Scp1中定义了两个肌动蛋白结合域，这两个结合域允许蛋白质在不需要二聚化的情况下结合和捆绑肌动蛋白。绿色荧光蛋白标记的Scp1突变体也表明，肌动蛋白定位并不需要假定的磷酸化位点Ser-185才能发挥作用。SCP1的缺失对细胞生长和形态几乎没有明显的影响。然而，我们发现scp1的缺失可以通过Sac6的上调来补偿。此外，在sac6缺失的情况下，Scp1水平升高。在缺乏Sac6或Scp1水平的情况下，代偿途径的存在表明维持足够的捆绑活性在细胞内是至关重要的。对内吞过程中皮质斑块组装和运动的分析揭示了先前未被发现的Scp1在斑块远离质膜的运动中的作用。此外，我们观察到在缺乏sac6和scp1的菌株中斑块寿命急剧增加，证明了肌动蛋白捆绑蛋白在内吞噬过程中发挥的核心作用。
The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process.