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

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 vht1⌬ mutants, also display aberrantly high expression of biotin-responsive genes. Like vht1⌬ cells, they have reduced levels of protein biotinylation, but unlike vht1⌬ 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.

The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in Saccharomyces cerevisiae

“…The same promoter fragment was fused to a full-length lacZ gene in a centromeric vector to give the reporter plasmid used in Fig. 5B (46). The yeast genomic library in the CEN-TRP1 vector pRS200 was described before (16) and obtained from the American Type Culture Collection (ATCC 77164).…”

“…Growth assays on agar plates were performed according to published methods (55). Biotinylated proteins were detected in whole-cell extracts using streptavidin-coupled peroxidase (strep-PO) as described previously (46). To remove strep-PO from desthiobiotinylated proteins, Western blots were incubated for 16 h in TBST (Tris-buffered saline with 0.05% Tween 20) buffer containing 2 mM desthiobiotin and 1% bovine serum albumin.…”

Effect of Biotin Treatment on Hepatic Gene Expression in Streptozotocin-Induced Diabetic Rats

“…Recently, Pirner and Stolz reported that, in yeast, the concentration of biotin in the culture medium affects the expression of the genes involved in biotin synthesis and transport. 31) The promoter region in these genes has a common nucleotide sequence, namely, the biotin response element (BRE), and a tentative transcription factor(s) is thought to bind to BRE in response to the intracellular biotin level to reduce the expression of these genes. We discovered in this experiment that the mRNA levels of at least 6 genes (PCK1, G6PC, HNF4, PGC-1, biotinidase, and PCCA) were significantly decreased by the biotin administration.…”

The Proline-Dependent Transcription Factor Put3 Regulates the Expression of the Riboflavin Transporter MCH5 in Saccharomyces cerevisiae

“…This plasmid was constructed in two steps. First, the MEL1 minimal promoter containing a fragment of the VHT1 pro-moter (Pirner and Stolz 2006) was ligated into the PstI and BamHI sites of YEplac195-GFP. Next, the VHT1 fragment was replaced with a PCR product containing the Put3 binding site (À501 to À486 bp relative to the start ATG of MCH5) along with 24 bp of upstream and 23 bp of downstream sequence.…”