Amino acids stimulate the endosome-to-golgi trafficking through ragulator and small GTPase Arl5
The endosome-to-Golgi trafficking pathway is an important post-Golgi recycling route. However, there is a lack of knowledge about the regulatory mechanisms behind intracellular membrane trafficking processes in response to extracellular signals. In this study, we found that nutrient starvation rever...
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2018
|
| Subjects: | |
| Online Access: | http://hdl.handle.net/10356/73545 |
| _version_ | 1826115590137315328 |
|---|---|
| author | Chen, Bing |
| author2 | Lu Lei |
| author_facet | Lu Lei Chen, Bing |
| author_sort | Chen, Bing |
| collection | NTU |
| description | The endosome-to-Golgi trafficking pathway is an important post-Golgi recycling route. However, there is a lack of knowledge about the regulatory mechanisms behind intracellular membrane trafficking processes in response to extracellular signals. In this study, we found that nutrient starvation reversibly caused the trans-Golgi network (TGN) membrane proteins, such as furin and CI-M6PR, translocate from the TGN to the endosomal pool. Using a series of CD8a tagged TGN membrane proteins as reporters, we demonstrated that nutrient could stimulate the endosome-to-Golgi trafficking. We found that amino acids (AAs), especially glutamine, but not growth factors or glucose, were the key factors regulating the endosome-to-Golgi trafficking in mammalian cells. Moreover, the stimulation effect of AAs on endosome-to-Golgi trafficking is probably ubiquitous, as it is observed in multiple cell lines. Thus, we made a novel discovery that the endosome-to-Golgi trafficking of cargos is inhibited and stimulated by the absence and presence, respectively, of AAs. Inspired by the mechanism of the AA-induced mTORC1 activation pathway, we hypothesized that the AA-stimulated endosome-to-Golgi trafficking pathway might share similar machinery. By selectively inhibiting or depleting each component of the AA-stimulated mTORC1 signaling pathway, it was revealed that SLC38A9, v-ATPase and Ragulator, but not Rag GTPases or mTORC1, are essential for AA-stimulated endosome-to-Golgi trafficking. To accomplish the delivery of cargos from endosomes to the Golgi, various factors, including tethering factors, SNAREs and the small GTPases from the Rab and Arf-like family, are involved. Arl5, an Arf-like family small GTPases, has been found to regulate the membrane trafficking between the endosome and the Golgi. There are three closely related paralogs of Arl5 in vertebrates – Arl5a, b and c, where Arl5a, Arl5b are the dominant ones. Endogenously and exogenously expressed Arl5a and Arl5b were found to localize in the Golgi, while human Arl5c did not display a Golgi localization. Using yeast two-hybrid, pull-down and immunoprecipitation assays, we found that Arl5 interacts with Lamtor1. Live-cell imaging revealed that Arl5b colocalizes with Lamtor1 at the endosome and lysosome. Furthermore, both Arl5 and its effector, the Golgi-associated retrograde protein complex (GARP), are required for AA-stimulated trafficking. We have therefore identified a mechanistic connection between nutrient signaling and the endosome-to-Golgi trafficking pathway, whereby SLC38A9 and v-ATPase sense AA-sufficiency. Moreover, the interaction between Lamtor1 and Arl5 might activate Arl5, which, together with its effector GARP, a tethering factor, likely facilitates the endosome-to-Golgi trafficking. |
| first_indexed | 2024-10-01T03:57:59Z |
| format | Thesis |
| id | ntu-10356/73545 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T03:57:59Z |
| publishDate | 2018 |
| record_format | dspace |
| spelling | ntu-10356/735452023-02-28T18:36:05Z Amino acids stimulate the endosome-to-golgi trafficking through ragulator and small GTPase Arl5 Chen, Bing Lu Lei School of Biological Sciences DRNTU::Science::Biological sciences The endosome-to-Golgi trafficking pathway is an important post-Golgi recycling route. However, there is a lack of knowledge about the regulatory mechanisms behind intracellular membrane trafficking processes in response to extracellular signals. In this study, we found that nutrient starvation reversibly caused the trans-Golgi network (TGN) membrane proteins, such as furin and CI-M6PR, translocate from the TGN to the endosomal pool. Using a series of CD8a tagged TGN membrane proteins as reporters, we demonstrated that nutrient could stimulate the endosome-to-Golgi trafficking. We found that amino acids (AAs), especially glutamine, but not growth factors or glucose, were the key factors regulating the endosome-to-Golgi trafficking in mammalian cells. Moreover, the stimulation effect of AAs on endosome-to-Golgi trafficking is probably ubiquitous, as it is observed in multiple cell lines. Thus, we made a novel discovery that the endosome-to-Golgi trafficking of cargos is inhibited and stimulated by the absence and presence, respectively, of AAs. Inspired by the mechanism of the AA-induced mTORC1 activation pathway, we hypothesized that the AA-stimulated endosome-to-Golgi trafficking pathway might share similar machinery. By selectively inhibiting or depleting each component of the AA-stimulated mTORC1 signaling pathway, it was revealed that SLC38A9, v-ATPase and Ragulator, but not Rag GTPases or mTORC1, are essential for AA-stimulated endosome-to-Golgi trafficking. To accomplish the delivery of cargos from endosomes to the Golgi, various factors, including tethering factors, SNAREs and the small GTPases from the Rab and Arf-like family, are involved. Arl5, an Arf-like family small GTPases, has been found to regulate the membrane trafficking between the endosome and the Golgi. There are three closely related paralogs of Arl5 in vertebrates – Arl5a, b and c, where Arl5a, Arl5b are the dominant ones. Endogenously and exogenously expressed Arl5a and Arl5b were found to localize in the Golgi, while human Arl5c did not display a Golgi localization. Using yeast two-hybrid, pull-down and immunoprecipitation assays, we found that Arl5 interacts with Lamtor1. Live-cell imaging revealed that Arl5b colocalizes with Lamtor1 at the endosome and lysosome. Furthermore, both Arl5 and its effector, the Golgi-associated retrograde protein complex (GARP), are required for AA-stimulated trafficking. We have therefore identified a mechanistic connection between nutrient signaling and the endosome-to-Golgi trafficking pathway, whereby SLC38A9 and v-ATPase sense AA-sufficiency. Moreover, the interaction between Lamtor1 and Arl5 might activate Arl5, which, together with its effector GARP, a tethering factor, likely facilitates the endosome-to-Golgi trafficking. Doctor of Philosophy (SBS) 2018-03-29T03:34:07Z 2018-03-29T03:34:07Z 2018 Thesis Chen, B. (2018). Amino acids stimulate the endosome-to-golgi trafficking through ragulator and small GTPase Arl5. Doctoral thesis, Nanyang Technological University, Singapore. http://hdl.handle.net/10356/73545 10.32657/10356/73545 en 149 p. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences Chen, Bing Amino acids stimulate the endosome-to-golgi trafficking through ragulator and small GTPase Arl5 |
| title | Amino acids stimulate the endosome-to-golgi trafficking through ragulator and small GTPase Arl5 |
| title_full | Amino acids stimulate the endosome-to-golgi trafficking through ragulator and small GTPase Arl5 |
| title_fullStr | Amino acids stimulate the endosome-to-golgi trafficking through ragulator and small GTPase Arl5 |
| title_full_unstemmed | Amino acids stimulate the endosome-to-golgi trafficking through ragulator and small GTPase Arl5 |
| title_short | Amino acids stimulate the endosome-to-golgi trafficking through ragulator and small GTPase Arl5 |
| title_sort | amino acids stimulate the endosome to golgi trafficking through ragulator and small gtpase arl5 |
| topic | DRNTU::Science::Biological sciences |
| url | http://hdl.handle.net/10356/73545 |
| work_keys_str_mv | AT chenbing aminoacidsstimulatetheendosometogolgitraffickingthroughragulatorandsmallgtpasearl5 |