OSBP-related protein 7 interacts with GATE-16 and negatively regulates GS28 protein stability.
OSBP-related protein 7 interacts with GATE-16 and negatively regulates GS28 protein stability.
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DOI：
10.1016/j.yexcr.2011.05.028
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发表时间：
2011-10
影响因子：
3.7
通讯作者：
Wenbin Zhong;You Zhou;Shiqian Li;Tianhong Zhou;Hongling Ma;Kunde Wei;Hongye Li;V. Olkkonen;Daoguang Yan
中科院分区：
文献类型：
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作者：
Wenbin Zhong;You Zhou;Shiqian Li;Tianhong Zhou;Hongling Ma;Kunde Wei;Hongye Li;V. Olkkonen;Daoguang Yan
ORP7是氧甾醇结合蛋白（OSBP）家族的一员，其功能尚不清楚。在本研究中，我们通过酵母双杂交筛选确定了ORP7的相互作用伙伴GATE-16，它(i)调节高尔基网28kDa （GS28）的功能和稳定性，（ii）在自噬体的生物发生中起作用。通过活细胞双分子荧光互补（BiFC）实验证实了这种相互作用。相互作用区分别位于ORP7的aa 1 ~ 142和GATE-16的aa 30 ~ 117内。293A细胞中ORP7敲低导致GS28蛋白增加40%，而ORP7过表达则相反（GS28蛋白减少25%）。我们证明ORP7对GS28的调控并不发生在转录水平，而是涉及到蛋白酶体上GS28的降解。缺少GATE-16结合区的截断ORP7没有影响GS28的稳定性，证明了观察到的效果的特异性。与ORP7过表达类似，25-羟基胆固醇（25-OH）处理细胞导致GS28不稳定，过量ORP7增强了这种不稳定，ORP7沉默抑制了这种不稳定。ORP7的过表达导致大多数细胞形成RFP-LC3阳性的空泡结构，从而代表自噬元件。此外，在液泡ORP7阳性元件中也发现了GATE-16，这表明过量的ORP7增加了自噬体中GATE-16的包裹。综上所述，我们的研究结果表明ORP7通过隔离GATE-16负性调节GS28蛋白的稳定性，并可能介导25-OH对GS28和高尔基功能的影响。
ORP7 is a member of oxysterol-binding protein (OSBP) family, the function of which has remained obscure. In this study, we identified by yeast two-hybrid screening an interaction partner of ORP7, GATE-16, which (i) regulates Golgi SNARE of 28kDa (GS28) function and stability, and (ii) plays a role in autophagosome biogenesis. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay in living cells. The interacting regions were delineated within aa 1–142 of ORP7 and aa 30–117 of GATE-16. ORP7 knock-down in 293A cells resulted in a 40% increase of GS28 protein while ORP7 overexpression had the opposite effect (25% decrease of GS28). We show evidence that the regulation of GS28 by ORP7 does not occur at the level of transcription, but involves degradation of GS28 on proteasomes. Truncated ORP7 that lacks the GATE-16 binding region failed to affect GS28 stability, evidencing for specificity of the observed effect. Similar to ORP7 overexpression, treatment of cells with 25-hydroxycholesterol (25-OH) resulted in GS28 destabilization, which was potentiated by excess ORP7 and inhibited by ORP7 silencing. Overexpression of ORP7 led in most cells to formation of vacuolar structures positive for RFP-LC3, thus representing autophagic elements. Also GATE-16 was found in the vacuolar ORP7-positive elements, suggesting that excess ORP7 increases entrapment of GATE-16 in autophagosomes. Taken together, our results suggest that ORP7 negatively regulates GS28 protein stability via sequestration of GATE-16, and may mediate the effect of 25-OH on GS28 and Golgi function.