The cytosolic tail of the Golgi apyrase Ynd1 mediates E4orf4-induced toxicity in Saccharomyces cerevisiae.

The adenovirus E4 open reading frame 4 (E4orf4) protein contributes to regulation of the progression of virus infection. When expressed individually, E4orf4 was shown to induce non-classical transformed cell-specific apoptosis in mammalian cells. At least some of the mechanisms underlying E4orf4-induced toxicity are conserved from yeast to mammals, including the requirement for an interaction of E4orf4 with protein phosphatase 2A (PP2A). A genetic screen in yeast revealed that the Golgi apyrase Ynd1 associates with E4orf4 and contributes to E4orf4-induced toxicity, independently of Ynd1 apyrase activity. Ynd1 and PP2A were shown to contribute additively to E4orf4-induced toxicity in yeast, and to interact genetically and physically. A mammalian orthologue of Ynd1 was shown to bind E4orf4 in mammalian cells, confirming the evolutionary conservation of this interaction. Here, we use mutation analysis to identify the cytosolic tail of Ynd1 as the protein domain required for mediation of the E4orf4 toxic signal and for the interaction with E4orf4. We also show that E4orf4 associates with cellular membranes in yeast and is localized at their cytoplasmic face. However, E4orf4 is membrane-associated even in the absence of Ynd1, suggesting that additional membrane proteins may mediate E4orf4 localization. Based on our results and on a previous report describing a collection of Ynd1 protein partners, we propose that the Ynd1 cytoplasmic tail acts as a scaffold, interacting with a multi-protein complex, whose targeting by E4orf4 leads to cell death.

YND1 interacts with CDC55 and is a novel mediator of E4orf4-induced toxicity.

Adenovirus E4orf4 (early region 4 open reading frame 4) protein induces protein phosphatase 2A-dependent non-classical apoptosis in mammalian cells and irreversible growth arrest in Saccharomyces cerevisiae. Oncogenic transformation sensitizes cells to E4orf4-induced cell death. To uncover additional components of the E4orf4 network required for induction of its unique mode of apoptosis, we used yeast genetics to select gene deletions conferring resistance to E4orf4. Deletion of YND1, encoding a yeast Golgi apyrase, conferred partial resistance to E4orf4. However, Ynd1p apyrase activity was not required for E4orf4-induced toxicity. Ynd1p and Cdc55p, the yeast protein phosphatase 2A-B subunit, contributed additively to E4orf4-induced toxicity. Furthermore, concomitant overexpression of one and deletion of the other was detrimental to yeast growth, demonstrating a functional interaction between the two proteins. YND1 and CDC55 also interacted genetically with CDC20 and CDH1/HCT1, encoding activating subunits of the anaphase-promoting complex/cyclosome. In addition to their functional interaction, Ynd1p and Cdc55p interacted physically, and this interaction was disrupted by E4orf4, which remained associated with both proteins. The results suggested that Ynd1p and Cdc55p share a common downstream target whose balanced modulation by the two E4orf4 partners is crucial to viability. Disruption of this balance by E4orf4 may lead to cell death. NTPDase-4/Lalp70/UDPase, the closest mammalian homologue of Ynd1p, associated with E4orf4 in mammalian cells, suggesting that the results in yeast are relevant to the mammalian system.