Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5.

Meng Shi,Lei Lu,Boon Kim Boh,Bing Chen,Hieng Chiong Tie,Yan Zhou,Geng Wu,Bamaprasad Dutta,Siu Kwan Sze,Divyanshu Mahajan

doi:10.1038/s41467-018-07444-y

Abstract

The endosome-to-Golgi or endocytic retrograde trafficking pathway is an important post-Golgi recycling route. Here we show that amino acids (AAs) can stimulate the retrograde trafficking and regulate the cell surface localization of certain Golgi membrane proteins. By testing components of the AA-stimulated mTORC1 signaling pathway, we demonstrate that SLC38A9, v-ATPase and Ragulator, but not Rag GTPases and mTORC1, are essential for the AA-stimulated trafficking. Arl5, an ARF-like family small GTPase, interacts with Ragulator in an AA-regulated manner and both Arl5 and its effector, the Golgi-associated retrograde protein complex (GARP), are required for the AA-stimulated trafficking. We have therefore identified a mechanistic connection between the nutrient signaling and the retrograde trafficking pathway, whereby SLC38A9 and v-ATPase sense AA-sufficiency and Ragulator might function as a guanine nucleotide exchange factor to activate Arl5, which, together with GARP, a tethering factor, probably facilitates the endosome-to-Golgi trafficking.

Highlights

The endosome-to-Golgi or endocytic retrograde trafficking pathway is an important postGolgi recycling route
To investigate if nutrient plays a role in the endocytic membrane trafficking, we compared the sub-cellular distribution of transGolgi network (TGN) membrane proteins in normal and starvation medium
In the complete medium (DMEM supplemented with 10% fetal bovine serum), endogenous furin mainly colocalized with Golgin-245, as expected (Fig. 1a, b)

Summary

Results

Starvation translocates TGN membrane proteins to endosomes. To investigate if nutrient plays a role in the endocytic membrane trafficking, we compared the sub-cellular distribution of TGN membrane proteins in normal and starvation medium. Starvation-induced change of localization was observed for other TGN membrane proteins such as endogenous CI-M6PR and overexpressed CD8a-CI-M6PR (Supplementary Fig. 1d-g). While the molecular and cellular mechanism and biological significances of the data require further investigation, we showed that cell surface presentation of Golgi membrane proteins can be regulated by AAs. Components regulating the AA-stimulated trafficking. Knockdown of Lamtor[1] inhibited the AA-stimulated reduction of CD8a-furin at the cell surface, suggesting an essential role of Ragulator-mediated endocytic trafficking in the surface localization of furin (Fig. 3i). Our results demonstrated that SLC38A9, v-ATPase, and Ragulator, but not Rag GTPases and mTORC1, are probably involved in the AA-stimulated endosome-to-Golgi trafficking. The resulting beads were incubated with HEK293T cell lysate expressing Lamtor1-GFP Both GMPPNP and GDP-loaded Arl5b pulled down Lamtor1-GFP (Fig. 4a). In contrast to mouse Arl5c, human Arl5c is significantly different from the rest paralogs as it does not have a typical G3 box

17 GST-Arl5b 48 35 GST

Discussion

Methods