oa The Role of C-Terminus Cytosolic Domain in the Mechanism of ORAI1 Trafficking and Internalization During Oocyte Maturation

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ influx pathway essential for many physiological functions and failure to maintain normal calcium homeostasis is one of the leading causes of cellular dysfunction in a wide variety of pathological conditions. Orai1, a key regulator of SOCE, constitutively recycles at steady state in the frog oocyte and internalizes into intracellular vesicular compartments during meiosis, leading to inactivation of SOCE. Such mechanism provides proper regulation of Ca²⁺ signaling in preparation for fertilization and embryonic development. 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.

We began the study of the molecular determinants of Orai1 trafficking in Xenopus oocytes by comparing the localization of multiple GFP-Orai1 C-terminal mutants 1-266, 1-275, and 1-285 intracellularly. Orai1 mutants 1-275 and 1-285 were both internalized during meiosis behaving similarly to WT, however, Orai1 1-266 was significantly enriched at the plasma membrane and did not internalize during oocyte maturation. To further map the region required for Orai1 internalization, we will generate specific mutants in the region 267-275 and evaluate the contribution of these signals in Orai1 internalization during meiosis. Mutants showing defect in internalization will be tested for phenotype rescue by co-injecting Orai1 with different potential candidates. These proteins include: 1) Rab5, a member of Rab family of GTPases has been shown to play a role as a regulator of intracellular trafficking during endocytosis. Previous data from our group has shown that Rab5 colocalizes with Orai1 in both oocytes and eggs. 2) Caveolin-1, a coat protein mediating caveolin-dependent endocytosis. 3) Flotilin-1, a membrane-associated protein involved in flotilin-dependent endocytosis.

As a second step in exploring the molecular and biochemical mechanisms underlying Orai1 internalization during meiosis, we will identify Orai1 associated proteins by co-immunoprecipitation of Orai1 complexes followed by quantitative proteomic analysis using dimethyl labeling coupled with tandem liquid chromatography-mass spectrometry (LC-MS). Candidates that are enriched in wild-type Orai1 immunoprecipitation but missing in internalization-deficient Orai1 mutants are suggested to play role in Orai1 endocytosis.

In conclusion, our results here suggest that residues 266–275 at C-terminus of Orai1 are essential for its internalization during meiosis. Using specific mutant(s) within this region, we will test the rescue of mutant(s) phenotype with potential candidates, and by quantitative proteomic analysis identify proteins associated with Orai1 and potentially important in its internalization.