Fraisier Vincent, Kasri Amal, Miserey-Lenkei Stéphanie, Sibarita Jean-Baptiste, Nair Deepak, Mayeux Adeline, Bardin Sabine, Toyoda Yusuke, Poser Ina, Poznyakovskiy Andrei, Goud Bruno, Hyman Anthony A, Dimitrov Ariane
UMR144, Institut Curie/ CNRS, Cell and Tissue Imaging Platform, Paris, France.
PLoS One. 2013 Dec 2;8(12):e82223. doi: 10.1371/journal.pone.0082223. eCollection 2013.
The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.
高尔基体是蛋白质翻译后修饰、分选和运输所必需的细胞内区室。它通过高尔基体有丝分裂检查点在有丝分裂进入过程中起关键作用。为了鉴定参与高尔基体有丝分裂检查点的新蛋白质,我们将有丝分裂表型的敲低筛选结果与定位筛选结果相结合。使用这种方法,我们鉴定出一种新的高尔基体蛋白C11ORF24(NP_071733.1)。我们表明,C11ORF24在N端有一个信号肽,在C端区域有一个跨膜结构域。C11ORF24定位于高尔基体和反式高尔基体网络。该蛋白的大部分存在于高尔基体腔中,而只有一小段尾巴延伸到细胞质中。这条细胞质尾巴在进化过程中高度保守。通过荧光漂白恢复实验,我们表明C11ORF24在高尔基体膜中的动态与该蛋白中跨膜结构域的存在一致。C11ORF24不仅存在于高尔基体上,还以pH敏感的方式通过内体循环到质膜。此外,通过视频显微镜研究,我们表明C11ORF24存在于运输中间体上,并与小GTP酶RAB6共定位,RAB6是一种参与从高尔基体到质膜的顺向运输的GTP酶。在我们的实验中,敲低C11ORF24不会导致有丝分裂表型或细胞内运输缺陷。总之,这些数据表明C11ORF24存在于高尔基体上,被运输到质膜,并通过RAB6阳性载体通过内体循环回来。