Structural and Functional Conservation Between Yeast and Human 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductases, the Rate-Limiting Enzyme of Sterol Biosynthesis

Abstract

The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.

Positive and Negative Transcriptional Control by Heme of Genes Encoding 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Saccharomyces cerevisiae

“…For construction of pJR436, a single-copy plasmid carrying an HMGI-lacZ fusion at the first ATG of the HMGJ open reading frame, the 0.25-kilobasepair (kbp) HindIII-BamHI fragment of plasmid pAB120 (obtained from K. Zaret and F. Sherman) that carries the CYCI transcription terminator (TERM; described in reference 41) (Fig. 1) was inserted into the HindIII-BamHI sites of the yeast centromere vector YCp5O (38) of plasmid pJR59, extending from nucleotide -957 of the HMGJ promoter to nucleotide +4 at the first ATG of the HMGJ open reading frame (5,44), was isolated and inserted into the HindIII-SphI sites of the polylinker of pEMBL18 (provided by R. Levis [12]) to generate plasmid pJR420. Next, the 3.0-kbp Sall fragment carrying the Escherichia coli lacZ gene was isolated from pMC1871 (9) and inserted into the Sall site of pJR420 to generate plasmid pJR425.…”

Protein Quality Control as a Strategy for Cellular Regulation: Lessons from Ubiquitin-Mediated Regulation of the Sterol Pathway

“…A multispanning N-terminal domain keeps the protein anchored in the ER, and a linker connects the membrane anchor to the highly conserved, soluble catalytic C-terminal region responsible for HMGR's essential enzyme activity in all eukaryotes and archebacteria. 27,28 It is the N-terminal multispanning domain that mediates regulated degradation of the HMGR molecule, allowing replacement of the C-terminal catalytic region with enzymatic or optical reporters while still preserving the regulated degradation of the molecule. The enzymes of ubiquitination.…”

Network analysis reveals the molecular bases of statin pleiotropy that vary with genetic background

“…This was cleanly illustrated with humanisation of yeast (i.e., expression of human genes in yeast) where only 20% amino acid identity was required for human genes to complement the deletion of orthologous yeast genes (Kachroo et al 2015). Relevant to this study, human HMGCR restored the viability of yeast lacking its two paralogue genes, HMG1 and HMG2 (Basson et al 1988). Indeed, many steps of the mevalonate pathway were originally elucidated in yeast (Bloch 1965; Hampton andRine 1994).…”