Defining the SPCA2C interactome identifies unique links to store-operated calcium entry

Calcium (Ca2+) is critical for normal cell function and several protein networks are required for Ca2+ signaling. In the pancreas, regulated changes in cytosolic Ca2+ allow for the exocytosis of zymogen granules and altered Ca2+ signaling underlies pancreatic pathologies. Previously, our laboratory showed a pancreas-specific isoform of secretory pathway Ca2+-ATPase 2 (SPCA2C) affects multiple pathways involved in Ca2+ homeostasis. The goal of this study was to define the SPCA2C interactome that contributes to these processes. Using proximity-dependent biotin identification, BioID, we expressed SPCA2C-BirA*HA in HEK293 cells with constitutive Orai1 expression. In silico modeling of SPCA2C showed a highly dynamic cytosolic C-terminus, revealing a putative site for interactions and was selected for BirA* fusion. 150 candidate SPCA2C interactors were identified. Gene Ontology and KEGG Pathway analyses supported localization of SPCA2C to the endoplasmic reticulum as well as function in Ca2+ signaling and suggested roles in vesicular transport. SPCA2C interactions with stromal interaction molecule 1 (STIM1), extended synaptotagmin-1 (ESYT1), and aspartate beta-hydroxylase (ASPH) were validated. Coiled-coil domain containing protein 47 (CCDC47) ranked as a high confidence SPCA2C-interactor which was confirmed through immunoprecipitation and co-localization with SPCA2C. CCDC47 interactions with SPCA2C, STIM1 and Orai1 were maintained following deletion of the CCDC47 luminal and cytosolic domains while deletion of only the coiled-coil domain altered CCDC47 localization and decreased interactions with STIM1 and Orai1. Overall, this study defines several novel protein interactions for SPCA2C and suggests it may be involved in affecting CCDC47, STIM1 and Orai1 function.