oa Identification Of Proteins Involved In Orai1 Trafficking By Mass Spectrometry-based Approach

Store-operated calcium entry (SOCE) is a ubiquitous Ca2+ influx pathway essential for many physiological functions. Dysregulation of SOCE causes disruption in Ca2+ homeostasis leading to cellular pathology of several diseases. Orai1, a key regulator of SOCE, constitutively recycles at steady state in the frog oocyte and internalizes into an intracellular vesicular compartments during meiosis, leading to inactivation of SOCE. Previous data showed a role for Orai1 C-terminus in its internalization during meiosis. However, the minimal region required for Orai1 internalization at steady state and during meiosis is not known. In this study we investigate the molecular determinants of Orai1 trafficking in Xenopus oocytes by comparing the localization of multiple Orai1 C-terminal mutants 1-266, 1-275, and 1-285 intracellularly. Orai1 mutants 1-275 and 1-285 were both present in endosomes and were internalized during meiosis behaving similarly to WT. However, Orai1 1-266 was enriched at the plasma membrane and showed decreased localization to the endosomal compartment at steady state. Furthermore, in contrast to wild-type Orai1, it was not internalized during oocyte maturation. To identify proteins interacting with Orai1 and involved in its trafficking, we co-immunoprecipitated Orai1 complexes from oocytes expressing WT Orai1 and Orai1 1-266 and analyzed complexes by mass spectrometry. Our preliminary data identified several candidate proteins co-immunoprecipitating with WT Orai1 but not with Orai1 1-266 mutant. To summarize, our results show that the region of Orai1 containing residues 267-275 is likely essential for Orai1 localization to endosomes suggesting a role for this domain in Orai1 intracellular distribution. Mass spectrometry analysis on co-immunoprecipitation of Orai1 WT identified several candidates that are missing in Orai1 1-266 co-IPs, suggesting a role for these candidates in Orai1 trafficking.