Biotin Sensing in Saccharomyces cerevisiae Is Mediated by a Conserved DNA Element and Requires the Activity of Biotin-Protein Ligase

Heike M. Pirner,Jürgen Stolz

doi:10.1074/jbc.m511075200

Abstract

Biotin is a water-soluble vitamin that functions as a prosthetic group in carboxylation reactions. In addition to its role as a cofactor, biotin has multiple roles in gene regulation. We analyzed biotin effects on gene expression in the yeast Saccharomyces cerevisiae and demonstrated by microarray, Northern, and Western analyses that all yeast genes encoding proteins involved in biotin metabolism are up-regulated following biotin depletion. Many of these genes contain a palindromic promoter element that is necessary and sufficient for mediating the biotin response and functions as an upstream-activating sequence. Mutants lacking the plasma membrane biotin transporter Vht1p display constitutively high expression levels of biotin-responsive genes. However, they react normally to biotin precursors that do not require Vht1p for uptake. The biotin-like effect of precursors with regard to gene expression requires their intracellular conversion to biotin. This demonstrates that Vht1p does not act as a sensor for biotin and that intracellular biotin is crucial for gene expression. Mutants with defects in biotin-protein ligase, similar to vht1delta mutants, also display aberrantly high expression of biotin-responsive genes. Like vht1delta cells, they have reduced levels of protein biotinylation, but unlike vht1delta mutants, they possess normal levels of free intracellular biotin. This indicates that free intracellular biotin is irrelevant for gene regulation and identifies biotin-protein ligase as an important element of the biotin-sensing pathway in yeast.

Highlights

Biotin, a water-soluble vitamin that functions as a prosthetic group of carboxylases, is an essential nutrient for most yeast species [5]
A Conserved DNA Element in the Promoters of Genes Involved in Biotin Metabolism—We have shown previously that S. cerevisiae and S. pombe possess plasma membrane biotin permeases that are transcriptionally regulated by the amount of biotin present in the growth medium [16, 20]
We provide evidence by microarray, Northern, Western, and reporter gene analyses that the S. cerevisiae genes involved in biotin metabolism are regulated by biotin

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains, Media, and Culture Conditions—W303-1A (MAT␣ ura ade trp his leu2-3,112 [29]) was used as a wild-type strain for most experiments. The kanMX-GAL1 promoter fusion was amplified with oligonucleotides that contained terminal regions of homology derived from BPL1 and mediated homologous recombination after transformation of the PCR product. The pyc2⌬::kanMX4 deletion in W303-1A was generated by transformation with a PCR fragment containing the pyc2⌬::kanMX4 allele from an EUROSCARF strain (Frankfurt/Main, Germany). The fragments of the VHT1 promoter depicted in Fig. 3 were cloned as PCR products with terminal XhoI or SalI sites into the unique XhoI site of pMEL-␤2, a centromeric yeast vector that contains the UAS-less MEL1 promoter fused to lacZ [37]. Determination of Intracellular Free Biotin—To quantify cellular free biotin, 30 –100 A600 units of cells were washed, resuspended in PBS containing 0.1% Tween 20 (PBST), boiled for 10 min at 95 °C, and centrifuged to remove cell fragments and denatured proteins. Probes were obtained by random labeling of PCR products derived from the coding regions of VHT1, BPL1, BIO2, BIO5, or ACT1 (encoding actin) with [␣-32P]dCTP

RESULTS

Intracellular biotin concentrations

Low extracellular biotin

Reporter gene activities of yeast strains lacking biotin proteins

DISCUSSION