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

Coimmunoprecipitation of E4orf4 with WT and mutant Ynd1 proteins.

<p>E4orf4 was immunoprecipitated from <i>ynd1Δ</i> yeast cells expressing the indicated Ynd1 proteins. Western blots of the immune complexes (IP) and input lysates (10% of IP) were stained with the indicated antibodies.</p

Transduction of E4orf4 toxicity by WT and mutant Ynd1 constructs.

<p><b>A</b>. <i>ynd1Δ</i> cells transformed with WT Ynd1 or mutant Ynd1 constructs in the presence or absence of E4orf4 were serially diluted (1∶5) and grown on glucose and galactose plates. <b>B</b>. <i>ynd1Δ</i> cells were transformed with WT Ynd1 or mutant constructs in the presence or absence of E4orf4. Yeast cell concentrations were equalized according to OD measurements, and equal amounts of yeast were grown on glucose and galactose plates. A quantitative comparison of the ability of WT and mutant Ynd1 constructs to mediate the E4orf4 toxic signal was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015539#s2" target="_blank">Materials and Methods</a> section, and reflects relative colony sizes in the presence and absence of E4orf4. The activity of WT Ynd1 was defined as 1. Three experiments were carried out containing 2 duplicates each. Error bars represent the standard error.</p

Subcellular localization of Ynd1 proteins and E4orf4.

<p><b>A</b>. <i>ynd1Δ</i> yeast cells expressing the indicated Ynd1 constructs were subjected to subcellular fractionation by differential centrifugation, and the presence of Ynd1 proteins in the various fractions was determined by a Western blot analysis. Rpb4 is a RNA polymerase II subunit which shuttles between the nucleus and the cytosol, and served here as a cytosolic fractionation marker. H: heavy membranes. L: light membranes. C: cytosol. <b>B</b>. <i>ynd1Δ</i> yeast cell extracts expressing vector control or WT Ynd1 together with E4orf4 were subjected to subcellular fractionation by differential centrifugation. Western blots were stained sequentially with antibodies recognizing tagged Ynd1, E4orf4, and the Tpd3 subunit of PP2A. <b>C</b>. Crude membranes were prepared from yeast cells expressing the indicated Ynd1 constructs or E4orf4, and were subjected to a Proteinase K protection assay in the presence or absence of Triton X-100. Proteins were analyzed by Western blots stained with the indicated antibodies. The Myc tag is fused to the amino terminus of Ynd1 constructs and the HA tag is fused to the cytosolic carboxyl tail. In this experiment, a longer exposure of the 518-Ynd1-containing blot allowed the presentation of a stronger signal in the blot to confirm that most of the protein was degraded by Proteinase K.</p