Abstract. Overproduction of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in yeast resulted in striking morphological effects on the structure of intracellular membranes. Specifically, stacks of paired membranes closely associated with the nuclear envelope were observed in strains that overproduced the HMG1 isozyme, one of two isozymes for HMG-CoA reductase in yeast. These nuclear-associated, paired membranes have been named "karmellae" In strains that overproduced the HMG1 isozyme, HMG-CoA reductase was present in the karmellar layers. At mitosis, karmellae were asymmetrically segregated: the mother cells inherited all of the karmellae and the daughter cells inherited none. A membranous structure of different morphology was occasionally found in cells that overproduced the HMG2 isozyme. These observations further establish the existence of cellular mechanisms that monitor the levels of membrane proteins and compensate for changes in these levels by inducing synthesis of particular types of membrane.

We have isolated two genes from yeast encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase [hydroxymethylglutaryl-coenzyme A reductase (NADPH); HMG-CoA reductase; EC 1.1.1.34], the rate-limiting enzyme of sterol biosynthesis. These genes, HMGI and HMG2, were identified by hybridization to a cDNA clone encoding hamster HMG-CoA reductase. DNA sequence analysis reveals homology between the amino acid sequence of the proteins encoded by the two yeast genes and the carboxyl-terminal half of the hamster protein. Cells containing mutant alleles of both HMGI and HMG2 are unable to undergo spore germination and vegetative growth. However, cells containing a mutant allele of either HMGI or HMG2 are viable but are more sensitive to compactin, a competitive inhibitor of HMG-CoA reductase, than are wild-type cells. Assays of HMG-CoA reductase activity in extracts from hmglP and hmg2-mutants indicate that HMG1 contributes at least 83% of the activity found in wild-type cells.

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-methylglutaryl coenzyme A reductase (HMG-CoA reductase) Analysis of sterol biosynthesis in the yeast Saccharomyces cerevisiae and in mammalian cells (7) led to the biochemical elucidation of this pathway, which produces all isoprene-containing compounds, including sterol, ubiquinone, dolichol, and isopentenylated adenosine in tRNA. The enzymatic steps of this pathway are highly conserved between S. cerevisiae and larger eucaryotes. Although the regulation of this pathway in yeast cells remains virtually unexplored, it has been examined extensively in mammalian cells. These studies have revealed that 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is the rate-limiting enzyme of the mammalian pathway.The rates of synthesis and degradation of mammalian HMG-CoA reductase are controlled by negative feedback regulation. Serum cholesterol in the form of low-density lipoprotein reduces the steady-state level of HMG-CoA reductase mRNA (14, 32) and also decreases the half-life of the protein (11,18). An unidentified nonsterol product of the pathway provides additional negative feedback regulation (9, 44). The response of HMG-CoA reductase to the diurnal cycle and to several hormones may be mediated by reversible phosphorylation of the enzyme (6, 55) and by allosteric effectors (49).Mammalian HMG-CoA reductase is an integral membrane glycoprotein of the endoplasmic reticulum (8, 34) and has also been identified in peroxisomes (29). A structural model for the endoplasmic reticulum form of hamster HMG-CoA reductase has been proposed which is based on secondarystructure predictions from the primary sequence and on proteolysis experiments (33). The NH2-terminal one-third of the protein (amino acids 1 to 339) is membrane bound and contains seven potential membrane-spanning domains and a single N-linked

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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.

The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase, HMGl and HMG2, each encode a functional isozyme. Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable. This paper describes a method of screening for recessive mutations in the HMGl gene, the gene encoding the majority of HMG-CoA reductase activity in the cell. This method should be applicable to the isolation of mutations in other cases of duplicated genes or duplicated functions in yeast. Four recessive point mutations were recovered in HMGl. These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group. Assays of HMGCoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMGl. Intragenic complementation occurred with partial restoration of enzymatic activity. HMGI was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2. A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.