Role of the rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function
Role of the rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function
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DOI：
10.1091/mbc.01-09-0463
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发表时间：
2002-08-01
影响因子：
3.3
通讯作者：
Kuno, T
中科院分区：
文献类型：
--
作者：
Cheng, H;Sugiura, R;Kuno, T
一个遗传筛选突变的合成致死与裂殖酵母钙调磷酸酶缺失导致识别Ypt 3，哺乳动物Rab 11 GTP结合蛋白的同源物。温度敏感的ypt 3-i5等位基因的突变体表现出多效性表型，如胞质分裂，细胞壁完整性和液泡融合的缺陷，这些都加剧了FK 506治疗，钙调磷酸酶的特异性抑制剂。绿色荧光蛋白（GFP）标记的Ypt 3显示细胞质染色，集中在生长部位，这种极化定位需要肌动蛋白细胞骨架。它也被检测为点状染色在肌动蛋白非依赖性的方式。电子显微镜显示，ypt 3-i5突变体积累异常的高尔基体样结构和假定的后高尔基体囊泡，这在限制性温度下显着增加。因此，在限制性温度条件下，ypt 3-i5突变体不能分泌与pho 1（+）前导肽融合的GFP。FK 506处理加重了异常高尔基体样结构的积累，并在允许的温度下引起SPL-GFP分泌的显著减少。这些结果表明，Ypt 3是胞吐途径的多个步骤所需的，其突变影响不同的细胞过程，并且钙调神经磷酸酶在功能上与这些细胞过程相关。
A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.