Organisation of human ER-exit sites: requirements for the localisation of Sec16 to transitional ER
Organisation of human ER-exit sites: requirements for the localisation of Sec16 to transitional ER
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DOI：
10.1242/jcs.044032
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发表时间：
2009-08-15
影响因子：
4
通讯作者：
Stephens, David J.
中科院分区：
文献类型：
--
作者：
Hughes, Helen;Budnik, Annika;Stephens, David J.
COPII复合物介导分泌物和相关机械选择性掺入到内质网膜上称为过渡性ER（tER）的专门位点处的出芽囊泡中。在这里，我们显示使用共聚焦显微镜，免疫金标记的冷冻切片和电子断层扫描，在人体细胞在稳定状态下，Sec 16定位到杯状结构的tER是空间上不同的定位其他COPII外套组件。我们发现，Sec 16定义了tER，而Sec 23-Sec 24和Sec 13-Sec 31定义了后来的结构，这些结构先于但不同于中间室。Sec 16的稳态定位与下游COPII组分Sec 23-Sec 24和Sec 13-Sec 31的定位无关。Sec 16在膜上和膜下循环的速率比其他COPII组分慢，具有更大的不动部分。我们定义了Sec 16 A的区域，该区域决定了其对tER膜的强大定位，并发现这需要一个高度带电的区域以及一个在物种之间显示出高序列同一性的中心结构域。Sec 16的中央保守结构域结合Sec 13，将tER膜定位与COPII囊泡形成连接起来。这些数据与Sec 16作为ERES COPII组装平台的模型一致。
The COPII complex mediates the selective incorporation of secretory cargo and relevant machinery into budding vesicles at specialised sites on the endoplasmic reticulum membrane called transitional ER (tER). Here, we show using confocal microscopy, immunogold labelling of ultrathin cryosections and electron tomography that in human cells at steady state, Sec16 localises to cup-like structures of tER that are spatially distinct from the localisation of other COPII coat components. We show that Sec16 defines the tER, whereas Sec23-Sec24 and Sec13-Sec31 define later structures that precede but are distinct from the intermediate compartment. Steady-state localisation of Sec16 is independent of the localisation of downstream COPII components Sec23-Sec24 and Sec13-Sec31. Sec16 cycles on and off the membrane at a slower rate than other COPII components with a greater immobile fraction. We define the region of Sec16A that dictates its robust localisation of tER membranes and find that this requires both a highly charged region as well as a central domain that shows high sequence identity between species. The central conserved domain of Sec16 binds to Sec13 linking tER membrane localisation with COPII vesicle formation. These data are consistent with a model where Sec16 acts as a platform for COPII assembly at ERES.