Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation
Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation
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DOI：
10.1016/j.yexcr.2005.08.014
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发表时间：
2005-11-15
影响因子：
3.7
通讯作者：
Sokabe, M
中科院分区：
文献类型：
--
作者：
Ozeki-Miyawaki, C;Moriya, Y;Sokabe, M
酿酒酵母 MIDI 基因产物 (Mid1) 是一种拉伸激活的 Ca2+ 通透性通道成分，是 Ca2+ 流入和维持暴露于交配信息素 α 因子的细胞活力所必需的。它由 548 个氨基酸 (aa) 残基组成，具有四个疏水片段：H1 (aa 2-22)、H2 (aa 92-111)、H3 (aa 337-356) 和 H4 (aa 366-388)。它还具有 16 个假定的 N-糖基化位点。在本研究中，与 GFP 缀合的连续截短的 Mid1 蛋白在酿酒酵母细胞中表达。包含从 H1 到 H3 的区域的截短蛋白 (Mid(1-360)-GFP) 正常定位于血浆和内质网 (ER) 膜中，并补充了 mid1 突变体的低活力和 Ca2+ 摄取活性，而包含从 H1 到 H2 的区域的 Mid(1-133)-GFP 则不然。缺少 H I 区域的 Mid1(Delta 3-22)-GFP 未能定位于质膜。膜分级分离显示，在a因子存在的情况下，仅含有H I的Mid1(1-22)-GFP定位于质膜中，这表明H I是负责a因子诱导的Mid1递送至质膜的信号序列。从 H1 到 H3 的区域是 Mid1 在血浆和 ER 膜中定位所必需的。最后，Mid1-GFP 向质膜的运输依赖于 Mid1 和转运蛋白 Sec12 的 N-糖基化。 (c) 2005 Elsevier Inc. 保留所有权利。
The Saccharomyces cerevisiae MIDI gene product (Mid1) is a stretch-activated Ca2+-permeable channel component required for Ca2+ influx and the maintenance of viability of cells exposed to the mating pheromone, alpha-factor. It is composed of 548-amino-acid (aa) residues with four hydrophobic segments, H1 (aa 2-22), H2 (aa 92-111), H3 (aa 337-356) and H4 (aa 366-388). It also has 16 putative N-glycosylation sites. In this study, sequentially truncated Mid1 proteins conjugated with GFP were expressed in S. cerevisiae cells. The truncated protein containing the region from H I to H3 (Mid(1-360)-GFP) localized normally in the plasma and endoplasmic reticulum (ER) membranes and complemented the low viability and Ca2+-uptake activity of the mid1 mutant, whereas Mid(1-133)-GFP containing the region from H1 to H2 did not. Mid1(Delta 3-22)-GFP lacking the H I region failed to localize in the plasma membrane. Membrane fractionation showed that Mid1(1-22)-GFP containing only H I localized in the plasma membrane in the presence of a-factor, suggesting that H I is a signal sequence responsible for the a-factor-induced Mid1 delivery to the plasma membrane. The region from H1 to H3 is required for the localization of Mid1 in the plasma and ER membranes. Finally, trafficking of Mid1-GFP to the plasma membrane was dependent on the N-glycosylation of Mid1 and the transporter protein Sec12. (c) 2005 Elsevier Inc. All rights reserved.