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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.

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.

Intracellular calcium ion is a universal messenger that plays a critical role in wide range of physiological functions and failure to maintain calcium homeostasis is linked to cellular dysfunctions in several diseases. Store operated calcium entry (SOCE) is a fundamental Ca2+ influx pathway at the cell membrane that is activated in response to intracellular store depletion. The SOCE machinery consists of the ER Ca2+ sensor STIM1, and Orai1 a Ca2+ selective channel at the plasma membrane (PM). Despite advancements in our understanding of the regulation of STIM1 and Orai1 very little is known about the biogenesis and trafficking of Orai1 and how that affects SOCE in mammalian cells. Here we describe the trafficking dynamics underlying Orai1subcelluar distribution at steady state and after store depletion. We show that at steady state 40% of total cellular Orai1 localizes to the PM of CHO cells. The other 60% of Orai1 localizes intracellularly. Time lapse imaging shows continuous shuttling of Orai1-positive vesicles between the PM and intracellular compartments. Quantitative measurements of the exocytosis and endocytosis rates at steady state show a rapid recycling rate of the protein between the intracellular pool and the plasma membrane. Following store depletion, ~80% of Orai1 is localized at PM. Interestingly, we localized Orai1 to novel sub-plasma membrane vesicular compartment of unknown molecular composition. To study the domain of Orai1 involved in its trafficking, we generated deletions of the N-(1-90) or C-(257-301)-terminus. Deletion of N-terminus did not change the surface to total distribution of Orai1, while C-terminus deletion significantly altered Orai1 distribution, with majority of Orai1-∆C localizes intracellularly. Similarly, deletion of residues 267-301 and residues 276-301 showed significantly decreased surface/total distribution compared to wild type (WT), but a less pronounced defect than Orai1-∆C. In contrast, 1-285 behaved like WT Orai1 showing that residues 286-301 are not required for Orai1 biogenesis and localization to the cell membrane. In conclusion, our data suggest that Orai1 recycles at high rate between PM and intracellular compartments at steady state and is enriched at PM after store depletion. Our results also suggest that the amino acid region (257-285) of Orai1 C-terminus is essential for its biogenesis and PM targeting.

Modulation of calcium homeostasis plays a key role in regulating fundamental cellular processes, including gene transcription, cell proliferation, differentiation, and apoptosis. Store-operated calcium entry (SOCE) is the major pathway in non-excitable cells for extracellular Ca2+ influx across the plasma membrane and is regulated by the content of the intracellular Ca2+ stores. Following store depletion, the Ca2+ sensor in ER, stromal interaction molecule 1 (STIM1) clusters in ER regions close to the plasma membrane and recruits Orai1, which is a Ca2+ selective channel resulting in SOCE. Despite the significant knowledge in understanding STIM1 subcellular distribution dynamics, little is known about the trafficking of Orai1. Our laboratory previously showed that in Xenopus leavis oocytes Orai1 shuttles between plasma membrane and endosomal compartments, and that it internalizes during meiosis. However, the subcellular distribution and trafficking of Orai1 in mammalian cells is not fully understood. In this study we show that at steady state around 46% of Orai1 is on the surface of the plasma membrane of CHO cells, while the remaining 54% localizes intracellularly, suggesting that Orai1 constitutively recycles between the two compartments. Our time lapse imaging shows continuous shuttling of Orai1 to and from the PM with an exocytosis rate T1/2 of around 5 minutes. We also measured the endocytic rate of Orai1 at 5% min-1. To identify the domain within Orai1 required for its trafficking, we generated deletions of either the N- or C-terminus of Orai1. Deletion of N-terminus does not affect Orai1 trafficking to the cell membrane in CHO cells. In contrast deletion of the whole C-terminus (257-301) significantly altered Orai1 trafficking to the cell membrane. Deletion of amino acids 285-301 of Orai1 C-terminus does not have any impact on Orai1 trafficking to PM, suggesting that the information necessary for Orai1 trafficking to PM is located in the segment 257-285 of Orai1 C-terminus. In this study we determined, for the first time, the percentage of Orai1 on PM of mammalian cells and showed through time lapse-imaging that it constitutively recycles between PM and intracellular compartments. Our results also suggest, for the first time, that the amino acid region (257-285) of Orai1 C-terminus is essential for its plasma membrane trafficking.