The asterisk denotes cells transformed with the plasmid pYES-TOPO+POF1 for overexpression of Pof1. Accordingly, to investigate the hypothesis that Pof1p is a cytidylyltransferase, the biological complementation assay of the PCT1 mutant strain was performed by overexpressing POF1 in cells challenged with heat shock stress because Δpct1 is sensitive to this stress [26]. Overexpression of POF1 was able to reverse the heat shock sensitivity of the Δpct1 strain (Figure 2B), suggesting that Pof1p and Pct1p share a common

function. Indeed, as Δpct1 cells, the Δpof1 strain was highly sensitive to heat shock. selleckchem Moreover, overexpression of POF1 also partially rescued the wild type phenotype in Δpof1 strain. Pure, recombinant Pof1p was obtained in the soluble fraction (Figure 3A), and Pof1p was assayed for phosphocholine or phosphoethanolamine cytidylyltransferase activities. Intriguingly, POF1 did not hydrolyse CTP as analyzed by thin layer chromatography (TLC), but instead it displayed ATPase activity (Figure 3B). The ATPase activity was independent of the presence of phospholipid precursors in the reaction media, indicating that Pof1p was not interacting with these substrates, at least when hydrolyzing ATP. The reaction products

were also analyzed by mass spectrometry, but no CDP-choline or CDP-ethanolamine could be detected (data not shown). Figure

PHA-848125 mw 3 Pof1p purification and activity analyses. (A) SDS-PAGE showing the purification of recombinant Pof1p obtained through metal affinity chromatography. Lane 1: molecular weight standard; subsequent lanes were different fractions obtained during the elution process. (B) Thin layer chromatography analyses to observe Pof1p ATP transferase activity; the controls were included to assay for alterations in CTP and ATP. See the Materials and Methods section for details. Since the ability of Pof1p to complement Pct1p function during heat shock is not related to CDP-choline activity, the hypothesis that Pof1p participates in some PLX3397 in vitro protein quality control was tested. Cells were submitted to ER stress, by exposing them to high concentrations of dithiothreitol (DTT) and tunicamycin (a protein Loperamide glycosylation inhibitor). Both agents are well known to provoke accumulation of unfolded proteins in the ER. Δpof1 cells displayed higher sensitivity to ER stress agents than wild-type cells and Δubc7 cells (mutant strain which lacks UBC7 gene which encodes ubiquitin conjugating enzyme involved in ERAD, a control cell line [27]) (Figure 4), suggesting that Pof1p is involved in UPR. Besides, Pof1p presented an ATPase-specific activity of 5 nmol of released phosphate per hour per μM enzyme (Figure 5A).