ARL5B, an ARF-like small GTPase localized to the trans-Golgi, is known for regulating endosome-Golgi trafficking and promoting the migration and invasion of breast cancer cells. Although a few interacting partners have been identified, the mechanism of the shuttling of ARL5B between the Golgi membrane and the cytosol is still obscure. Here, using GFP-binding protein (GBP) pull-down followed by mass spectrometry, we identified heat shock cognate protein (HSC70) as an additional interacting partner of ARL5B. Our pull-down and isothermal titration calorimetry (ITC)-based studies suggested that HSC70 binds to ARL5B in an ADP-dependent manner. Additionally, we showed that the N-terminal helix and the nucleotide status of ARL5B contribute to its recognition by HSC70. The confocal microscopy and cell fractionation studies in MDA-MB-231 breast cancer cells revealed that the depletion of HSC70 reduces the localization of ARL5B to the Golgi. Using in vitro reconstitution approach, we provide evidence that HSC70 fine-tunes the association of ARL5B with Golgi membrane. Finally, we demonstrated that the interaction between ARL5B and HSC70 is important for the localization of cation independent mannose-6-phosphate receptor (CIMPR) at Golgi. Collectively, we propose a mechanism by which HSC70, a constitutively expressed chaperone, modulates the Golgi association of ARL5B, which in turn has implications for the Golgi-associated functions of this GTPase.

中文翻译：

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与热休克同源蛋白 HSC70 结合可微调小 GTP 酶 ARL5B 的高尔基体关联。

ARL5B 是一种定位于跨高尔基体的类似 ARF 的小 GTP 酶，已知可调节内体-高尔基体运输并促进乳腺癌细胞的迁移和侵袭。尽管已经确定了一些相互作用的伙伴，但 ARL5B 在高尔基体膜和细胞质之间穿梭的机制仍然不清楚。在这里，使用 GFP 结合蛋白 (GBP) 下拉和质谱分析，我们确定了热休克同源蛋白 (HSC70) 作为 ARL5B 的另一个相互作用伙伴。我们基于 Pull-down 和等温滴定量热法 (ITC) 的研究表明，HSC70 以 ADP 依赖性方式与 ARL5B 结合。此外，我们还发现 ARL5B 的 N 端螺旋和核苷酸状态有助于 HSC70 对其进行识别。MDA-MB-231 乳腺癌细胞的共聚焦显微镜和细胞分级研究表明，HSC70 的消耗减少了 ARL5B 在高尔基体的定位。使用体外重建方法，我们提供了 HSC70 微调 ARL5B 与高尔基膜关联的证据。最后，我们证明了 ARL5B 和 HSC70 之间的相互作用对于阳离子独立的 6-磷酸甘露糖受体 (CIMPR) 在高尔基体的定位很重要。总的来说，我们提出了一种机制，HSC70（一种组成型表达的伴侣）通过该机制调节 ARL5B 的高尔基体关联，这反过来又对该 GTP 酶的高尔基体相关功能产生影响。