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Alternative translation initiation gives rise to two isoforms of Orai1 with distinct plasma membrane mobilities.

ABSTRACT: Store-operated calcium entry is an almost ubiquitous signaling pathway in eukaryotic cells. The plasma membrane store-operated channels are comprised of subunits of the recently discovered Orai proteins, the major one being Orai1.We have discovered that native Orai1, as well as expressed Orai1, exists in two forms in similar quantities: a longer form (Orai1?) of approximately 33 kDa, and a shorter form (Orai1?) of approximately 23 kDa. The Orai1? form arises from alternative translation initiation from a methionine at position 64, and possibly also 71, in the longer Orai1? form. In the sequence upstream of the initiation site of Orai1?, there is a poly-arginine sequence previously suggested to be involved in interaction of Orai1 with plasma membrane phosphatidylinositol-4,5-bisphosphate. The loss of this phospholipid binding domain would be expected to influence the mobility of Orai1 protein in the plasma membrane. Indeed, experiments utilizing fluorescence recovery after photobleaching (FRAP) revealed that the recovery half-time for Orai1? was significantly faster than for Orai1?. Since Orai1 must diffuse to sites of interaction with the Ca(2+) sensor, STIM1, these two mobilities might provide for efficient recruitment of Orai1 subunits to sites of store-operated Ca(2+) entry during agonist-induced Ca(2+) signaling.

SUBMITTER: Fukushima M

PROVIDER: S-EPMC3516440 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Alternative translation initiation gives rise to two isoforms of Orai1 with distinct plasma membrane mobilities.

Fukushima Miwako M Tomita Takuro T Janoshazi Agnes A Putney James W JW

Journal of cell science 20120528 Pt 18

Store-operated calcium entry is an almost ubiquitous signaling pathway in eukaryotic cells. The plasma membrane store-operated channels are comprised of subunits of the recently discovered Orai proteins, the major one being Orai1.We have discovered that native Orai1, as well as expressed Orai1, exists in two forms in similar quantities: a longer form (Orai1α) of approximately 33 kDa, and a shorter form (Orai1β) of approximately 23 kDa. The Orai1β form arises from alternative translation initiati ...[more]

PMID: 22641696

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Project description:Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5' end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5' sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5' untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.

Dataset Information

Alternative translation initiation gives rise to two isoforms of Orai1 with distinct plasma membrane mobilities.

Publications

Alternative translation initiation gives rise to two isoforms of Orai1 with distinct plasma membrane mobilities.

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