Differential Regulation of Ceramide Synthase Components LAC1 and LAG1 in Saccharomyces cerevisiae

Northern blot analysis of LAC1 and LAG1 transcription. Total RNAs were prepared from the indicated strains, electrophoresed through formaldehyde-agarose gels, and transferred to a nylon membrane. 18S RNA was visualized by staining to ensure equal loading, and LAC1 or LAG1 transcripts were detected by probing the blot with ³²P-labeled DNA fragments from each coding region. (A) LAC1 mRNA levels are shown with relevant genetic backgrounds indicated. Plasmids carrying wild-type PDR1 (pPDR1), PDR3 (pPDR3), or hyperactive PDR3 (pPDR3-11) are listed along with the empty vector control (pRS315). (B) LAG1 mRNA levels are shown.

Western blot analysis of HA-tagged Lac1p and Lag1p. Whole-cell protein extracts were prepared from the indicated strains, and equal amounts of each extract were electrophoresed by SDS-PAGE. Each strain contained a low-copy-number plasmid expressing a 3× HA-tagged form of either Lac1p (top) or Lag1p (bottom). Where indicated, a low-copy-number plasmid carrying hyperactive PDR3 (PDR3-11) or the empty vector (pRS315) was also present. After electrophoresis, the proteins were transferred to nitrocellulose and the membrane was probed with an anti-HA antibody. A monoclonal antibody directed against the vacuolar membrane protein Vph1p was used to ensure equal loading and transfer.

A single PDRE mediates Pdr pathway regulation of LAC1. Low-copy-number plasmids carrying either a wild-type LAC1 promoter fusion to lacZ (LAC1-lacZ) or a mutant promoter lacking the PDRE (mPDRE-LAC1-lacZ) were introduced into the strains listed at the bottom of the figure (relevant genetic markers are shown). An empty vector plasmid (pRS315) or the same vector carrying the hyperactive PDR3-11 allele were present where indicated. Transformants were grown to mid-log phase, and β-galactosidase activities were determined as described in Materials and Methods.

Pdr1p and Pdr3p bind to LAC1 PDRE in vitro. DNA fragments containing the PDRE region from either the wild-type LAC1 promoter (wt) or the site-directed mutant form (mPDRE) were radiolabeled with ³²P as described in Materials and Methods. Each probe was incubated with no added proteins, with protein extracts from bacterial cells carrying an empty expression vector (vector only), or with the same vector expressing the DNA binding domain of Pdr1p or Pdr3p. Protein-DNA complexes were then treated with DNase I followed by electrophoresis through denaturing acrylamide-urea gels as described previously (32). The location of the PDRE (indicated to the left) was determined by comparison to a Maxam-Gilbert chemical cleavage reaction and restriction digestion (not shown).

Deletion mapping of the LAC1 promoter. A series of 5′ and internal promoter deletion derivatives of the LAC1-lacZ plasmid were generated. The solid lines indicate the extent of the promoter that remains in each construct. The name of each mutant construct is shown on the left side of the figure. A line drawing of the wild-type LAC1 promoter region is shown at the top with numbers referring to distances from the ATG codon. The dark gray boxes show the location of the putative Cbf1p binding sites, the white box corresponds to the PDRE, and the light gray box denotes the Rox1p binding site. The PDRE was altered by a site-directed mutation in pMK010303 and was precisely deleted from pMK0104-4. Each plasmid was transformed into isogenic wild-type (BY4742) or rox1Δ (BY4742 rox1Δ) cells, and β-galactosidase activities were assayed as described in the text.

Recombinant Rox1p binds to an element in the LAC1 promoter. DNase I protection analysis was used as described in the text. A purine-specific chemical cleavage reaction (AG) was used to locate the Rox1p binding site (shown to the left). DNase I digestion was carried out in the absence of added protein (no protein) or with increasing amounts of Rox1p (indicated at the top).

LAC1 and LAG1 expression control by Cbf1p and Rox1p. Low-copy-number plasmids containing the lacZ fusion genes listed were introduced into the indicated isogenic strains. Transformants were grown to mid-log phase and β-galactosidase activities were determined. (A) Expression of LAC1-lacZ fusion plasmids plotted on the same scale for comparison. (B) Expression of LAG1-lacZ plotted on an expanded scale to emphasize the observed change in gene expression.

LCB2 and SUR2 response to elevated Pdr pathway activity and rox1Δ. LCB2-lacZ and SUR2-lacZ fusions carried on low-copy-number plasmids were introduced into isogenic wild-type or rox1Δ strains along with an empty vector (pRS315) or the same plasmid carrying the hyperactive PDR3-11 allele. Transformants were grown and assayed for β-galactosidase activity as described in the text.