Differential distribution of dynamin isoforms in mammalian cells
Differential distribution of dynamin isoforms in mammalian cells
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DOI：
10.1091/mbc.9.9.2595
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发表时间：
1998-09-01
影响因子：
3.3
通讯作者：
McNiven, MA
中科院分区：
文献类型：
--
作者：
Cao, H;Garcia, F;McNiven, MA
发动蛋白是在受体介导的内吞作用期间对网格蛋白包被的囊泡形成必不可少的100-kDa GTP酶。迄今为止，已经鉴定出三种不同的发动蛋白基因，每种基因表达至少四种不同的可变剪接形式。目前，尚不清楚这些不同的发动蛋白基因产物是否执行不同或冗余的细胞功能。因此，本研究的重点是从大鼠组织中鉴定发动蛋白的其他剪接变体，并确定发动蛋白家族成员在培养的大鼠上皮细胞模型（克隆9细胞）中的分布。经过长距离逆转录（RT）-PCR的mRNA从不同的大鼠组织，全长cDNA编码不同的发动蛋白亚型进行测序，并揭示了四个额外的剪接变体发动蛋白I和九个发动蛋白III。因此，在大鼠组织中，总共有至少25种不同的mRNA由三种发动蛋白基因产生。随后，我们产生了稳定转染的克隆9细胞，其表达用绿色荧光蛋白标记的六种不同剪接形式的全长cDNA。这些转染细胞的共聚焦或荧光显微镜显示，许多发动蛋白与不同的膜室，其中包括网格蛋白包被的坑在质膜和高尔基体，和几个未定义的囊泡群体。这些结果表明，发动蛋白家族是更广泛的比原来预测的，并建议，不同的发动蛋白蛋白定位于不同的细胞质或膜隔室。
Dynamins are 100-kDa GTPases that are essential for clathrin-coated vesicle formation during receptor-mediated endocytosis. To date, three different dynamin genes have been identified, with each gene expressing at least four different alternatively spliced forms. Currently, it is unclear whether these different dynamin gene products perform distinct or redundant cellular functions. Therefore, the focus of this study was to identify additional spliced variants of dynamin from rat tissues and to define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells). After long-distance reverse transcription (RT)-PCR of mRNA from different rat tissues, the full-length cDNAs encoding the different dynamin isoforms were sequenced and revealed four additional spliced variants for dynamin I and nine for dynamin III. Thus, in rat tissues there are a total of at least 25 different mRNAs produced from the three dynamin genes. Subsequently, we generated stably transfected Clone 9 cells expressing full-length cDNAs of six different spliced forms tagged with green fluorescent protein. Confocal or fluorescence microscopy of these transfected cells revealed that many of the dynamin proteins associate with distinct membrane compartments, which include clathrin-coated pits at the plasma membrane and the Golgi apparatus, and several undefined vesicle populations. These results indicate that the dynamin family is more extensive than was originally predicted and suggest that the different dynamin proteins are localized to distinct cytoplasmic or membrane compartments.