PAQR3 Regulates Endoplasmic Reticulum-to-Golgi Trafficking of COPII Vesicle via Interaction with Sec13/Sec31 Coat Proteins

摘要

class="head no_bottom_margin" id="sec1title">IntroductionCellular homeostasis relies on the coordinated interactions of proteins and the correct localization of those proteins to specific subcellular compartments. Mapping these network and subcellular proteome associated with a protein is critical for revealing its biological functions. There have been several experimental approaches established to globally define protein interactions and localization. Proximity labeling is one of the most effective approaches that can obtain spatially resolved proteomic maps of specific proteins and compartments within living cells (). Proximity labeling relies on a engineered ascorbate peroxidase (APEX), which is capable of generating free biotinyl radicals to enable a rapid and spatially restricted labeling of proteins in the vicinity of the enzyme (, , ). APEX is a first-generation enzyme with triple mutations, whereas APEX2 is the improved second-generation enzyme with one additional mutation (, , , , ). The irreversible conjugation of biotin catalyzed by APEX2 enables the capture of labeled proteins for proteomic analysis.PAQR3, also named as RKTG for Raf kinase trapping to Golgi, is a Golgi-resident seven-transmembrane protein and plays a key role in maintaining cellular and physiological homeostasis (, ). As a member of the progestin and AdipoQ receptor (PAQR) family, PAQR3 is conserved during evolution (). Previous studies have demonstrated that PAQR3 is a multifunctional protein. PAQR3 suppresses cell growth and tumorigenesis mainly through negative regulation of Ras-Raf-MEK-ERK and PI3K-AKT signaling pathways (, , ). Both human and mouse studies have indicated that PAQR3 is a tumor suppressor that has an inhibitory function in many types of tumors (href="#bib20" rid="bib20" class=" bibr popnode">Jiang et al., 2011, href="#bib26" rid="bib26" class=" bibr popnode">Ling et al., 2014, href="#bib50" rid="bib50" class=" bibr popnode">Wang et al., 2012, href="#bib54" rid="bib54" class=" bibr popnode">Xie et al., 2008, href="#bib59" rid="bib59" class=" bibr popnode">Zhang et al., 2010). PAQR3 can regulate Golgi-to-plasma membrane (PM) transport via the Gβγ–PKD signaling pathway (href="#bib16" rid="bib16" class=" bibr popnode">Hewavitharana and Wedegaertner, 2015). Additionally, PAQR3 can control autophagy on glucose and amino acids starvation by integrating AMPK signaling to improve ATG14L-associated class III PI3K activity and by modulating mTOC1 activity (href="#bib49" rid="bib49" class=" bibr popnode">Wang et al., 2017, href="#bib56" rid="bib56" class=" bibr popnode">Xu et al., 2016). PAQR3 also plays a critical role in DNA damage repair by affecting the function of RAD23B-XPC (href="#bib58" rid="bib58" class=" bibr popnode">You et al., 2017). Moreover, PAQR3 has an important effect on hepatic lipid catabolism by promoting PPARα ubiquitination through the E3 ubiquitin ligase HUWE1 (href="#bib60" rid="bib60" class=" bibr popnode">Zhao et al., 2018).In this study, we applied the proximity labeling strategy to further investigate the interaction networks of PAQR3. We constructed APEX2-fused PAQR3 to label and capture proteins adjacent to PAQR3 in live cells. We then characterized the captured proteins by mass spectrum analysis. A series of detailed analyses led us to discover that PAQR3 can regulate ER-to-Golgi transport through interacting with COPII components Sec13/Sec31A, thereby extending the understanding about the biological functions of PAQR3, a key protein in maintaining cellular homeostasis.