Deletion of the single yeast lipin gene (<i>PAH1</i>) enhances TBSV repRNA accumulation.

Abstract

<p>(A) The role of Pah1p phosphatidate phosphatase and Dgk1p diacylglycerol kinase in lipid synthesis in yeast. To convert phosphatidic acid (PA) to diacylglycerol (DAG), Pah1p is dephosphorylated (activated) by the ER-localized Nem1p/Spo7p complex. (B) Top panel: Replication of the TBSV repRNA in wt and <i>pah1Δ</i> yeast was measured by Northern blotting 24 h after initiation of TBSV replication. Yeast co-expressed the TBSV (lanes 1–6) and the CNV (lanes 7–12) p33 and p92 replication proteins. The accumulation level of repRNA was normalized based on the ribosomal (r)RNA. Each sample is obtained from different yeast colonies. Middle and bottom panels: The accumulation levels of FLAG-p92 and 6×His-p33 were tested by Western blotting. Each experiment was repeated. (C–D) Expression of wt Pah1p and a phosphorylation deficient, constitutively active Pah1p, called Pah1-7A, which contains alanine substitutions for all seven phosphorylation sites, reduces TBSV replication in <i>pah1Δ</i> and wt yeasts. Northern blotting was done as in panel B. (E) Stimulatory effect of deletion of <i>NEM1</i> and <i>SPO7</i>, which form the dephosphorylation complex in the ER membrane, on TBSV repRNA accumulation is shown by Northern blotting. Note that Nem1p and Spo7p are required to dephosphorylate Pah1p, leading to the activation and relocalization of Pah1p from the cytosol to the ER membrane.</p

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