S. cerevisiae requires three sulfur-containing amino acids to live. In addition to Met
and Cys, which are incorporated into cellular proteins, cells also require S-adenosylmethionine (AdoMet), which supplies activated methyl groups for many methylation reactions. The consensus view of the synthesis of these three amino acids on the previous page is now well- supported by biochemical and genetic evidence from many laboratories (reviewed in Thomas
& Surdin-Kerjan, 1992). The gene-enzyme relationships could not be definitively established until the development of molecular cloning and DNA sequencing techniques, which enabled investigators to use plasmid complementation to test gene function directly. In these experiments, investigators constructed plasmids with wild type MET, CYS or SAM genes, which were transformed into mutant strains. Transformed strains were only able to survive when the plasmid contained the wild type allele of the inactivated gene in the mutant. (You will use plasmid complementation in this class to confirm the identification of your strains and plasmids.)
Most of the genes that we will be working with this semester encode enzymes that catalyze an interconversion of one sulfur-containing molecule to a second sulfur-containing molecule. Other MET genes encode enzymes that do not directly participate in the synthesis of sulfur amino acids, but catalyze the synthesis of a cofactor or a methyl donor required for synthesis of sulfur amino acids. In the brief description below, we will follow the progress of a sulfur atom from inorganic sulfate through its conversion to Met, Cys or AdoMet.
Sulfate assimilation involves sulfur activation and reduction to sulfide
The early steps of the pathway, which encompasses the reactions involved in the conversion of sulfate to sulfide, comprise the sulfate assimilation pathway. Sulfate ions are the source of most sulfur in biological molecules, but considerable metabolic energy is required to activate sulfate from its +6 oxidation state and to convert it into sulfide, which has a -2 oxidation state. The enzymes responsible for sulfate assimilation are widely distributed in microorganisms and plants. In S. cerevisiae, sulfate is first activated by ATP sulfurylase, or Met3p, to form 5’-adenylylsulfate (APS). APS is then phosphorylated by Met14p, or APS kinase, forming 3’-phospho-5’-adenylylsulfate (PAPS). PAPS is an interesting molecule, since it contains an activated sulfur atom that can be used for a variety of sulfur transfer reactions. In mammals, PAPS in used for a variety of sulfation reactions in the Golgi, where the acceptors include lipids, proteins and a variety of small molecules. (Interestingly, APS kinase is the only yeast enzyme involved in sulfate assimilation with homologs in mammals.)
The final two steps in sulfate assimilation are NADPH-dependent reduction reactions. PAPS reductase, or Met16p, catalyzes the first reaction, which adds two electrons to the sulfur atom. The final 6-electron reduction is catalyzed by sulfite reductase. Sulfite reductase is a complex metalloenzyme containing two Met5p and two Met10p subunits as well as multiple prosthetic groups, including siroheme, that participate in electron transfer. (A prosthetic group is a metal ion or organic molecule that is covalently bound to an enzyme and essential for its activity.) In yeast, siroheme is synthesized in a series of reactions catalyzed by Met1p and Met8p. Siroheme synthesis is not formally considered to be part of the sulfate assimilation pathway, but its function is critical for the assembly of functional sulfite reductase.
Homocysteine synthesis and transsulfuration
In the next step of Met and Cys biosynthesis, sulfide becomes incorporated into the amino acid homocysteine (Hcy). Hcy sits at the branch point between several pathways in yeast. The amino acid backbone of Hcy ultimately derives from aspartic acid, which has been converted in
a series of steps to homoserine. (Note: “homo” amino acids have an extra carbon atom in their side chains compared to the namesakes without the prefix.) Met2p activates the homoserine
in an acetylation reaction that uses acetyl-CoA. Met17p, also known as either homocysteine synthase or O-acetyl homoserine sulfhydryase, then catalyzes the reaction of O-acetylhomoserine with sulfide to form Hcy.
In yeast, Hcy serves as the precursor for either Cys or Met. The pathway connecting Hcy and Cys is referred to as the transsulfuration pathway. Transsulfuration provides S. cerevisiae with unusual flexibility with respect to sulfur sources. Four different gene products are involved in the conversion of Hcy to Cys and vice versa, using cystathionine (below) as a common intermediate. Str2p catalyzes cystathionine synthesis from Cys and O-acetylhomoserine, the product of the reaction catalyzed by Met2p. In the opposite pathway, Cys4p (aka Str4p) catalyzes cystationine synthesis from Hcy and Ser. The four genes in the sulfur transfer pathway show different patterns of evolutionary conservation. For example, E. coli is unable to synthesize Cys from Met, while mammals are unable to synthesize Met from Cys.
Cystathionine is the intermediate for transsulfuration reactions. Enzymes in the S. cerevisiae transsulfurationpathway are encoded by the STR1-STR4 genes. Str2pand Str1p (Cys3p) catalyze the synthesis and hydrolysis, respectively, of the cystathionine S-Cg bond. Str3p and Str4p (Cys4p) catalyze the synthesis and hydrolysis, respectively, of the cystathionine S-Cb bond.
Methionine and AdoMet are formed during the methyl cycle
Hcy is also the starting point of a cycle that produces Met and AdoMet. The cycle begins as Met6p catalyzes the conversion of Hcy to Met, using an unusual methyl donor, polyglutamyl 5-methyl-tetrahydrofolate (THF). The MET13 and MET7 genes encode the enzymes that catalyze the last two steps in the synthesis of polyglutamyl 5-methyl-THF, which accounts for their inability of met7 and met13 cells to synthesize methionine.
As you might expect, most methionine is used for protein synthesis in cells, but an appreciable amount is converted to the high energy methyl donor, AdoMet, by two nearly identical AdoMet synthases, Sam1p and Sam2p. S. cerevisiae is able to synthesize large quantities of AdoMet, which is either used for transmethylation reactions or stored in its vacuole. (In fact, yeast is the source for most commercially-produced AdoMet.) Multiple yeast methyltranferases catalyze the transfer of methyl groups from AdoMet to hundreds of different substrates, which include nucleotide bases and sugars in DNA and RNA, various amino acid side chains in proteins, lipids, small molecules, and more. Each transmethylation reaction generates one molecule of S-adenosylhomocysteine (AdoHcy), which is hydrolyzed to adenosine and Hcy by Sah1p, completing the methyl cycle.
We will not be studying the enzymes involved in the methyl cycle in this class, but
it is important to appreciate their importance to cell survival. The amino acid sequences of Sam1p and Sam2p are 93% identical, which is far higher than other proteins that have arisen by gene duplication in S. cerevisiae. This redundancy provides a buffer against loss of either function. Cells with a mutation in either the SAM1 or SAM2 gene are able to survive, but cells with mutations in both genes are unable to survive. Similarly, the SAH1 gene is one of the few essential genes in S. cerevisiae, probably because the build-up of AdoHcy would inhibit many methyltransferase reactions.
Mutations disrupt biochemical pathways
The met mutants that you are analyzing are unable to catalyze one of the reactions required for sulfur amino acid synthesis. In this lab, you will use selective and differential media to determine which genes have been inactivated in your strains. Think of each mutation as erasing one of the arrows shown in the sulfur amino acid pathway. Our selective media contain a variety of sulfur sources. Depending on the position of the met mutation relative to the sulfur source, the strain may or may not be able to synthesize the sulfur amino acids.
You will also be using the differential medium, BiGGY agar to distinguish yeast strains
by the quantity of hydrogen sulfide that they produce. This is because BiGGY contains bismuth, which reacts with sulfide to form a brownish to black precipitate. All strains are expected to grow on BiGGY, since it contains yeast extract, which is a source of methionine. BiGGY also contains sulfite, rather than sulfate, as the primary sulfur source. Locate the positions of your mutated genes in the pathway relative to sulfide and sulfite. Mutations in genes upstream of sulfide should produce lighter colonies, since less sulfide will be produced. Of these, mutations that prevent sulfite reduction should produce the lightest colonies. Mutations in genes downstream of sulfide should produce darker colonies, because the strains will be unable to metabolize sulfide.
In making your predictions for this experiment, you may find this analogy useful: A metabolic pathway is not unlike a one-way (due to energetic considerations, most metabolic pathways are unidirectional) highway with a series of bridges connecting islands. (The islands have different energy levels.) The cars passing along the highway are the molecules that are being converted from one form to another. When a car reaches the next island in the pathway, its color changes because it has been converted into a different molecule. The bridges are the enzymes
in the pathway. They facilitate the passage of the cars, because they catalyze the reactions that convert one molecule to the next. If a mutation occurs in a gene that encodes a particular enzyme, that particular bridge falls down. Cars begin to pile up before the broken bridge, and very few cars would be found on islands past the broken bridge. In some cases, there may be an alternative route, or salvage pathway, but this is usually a less efficient route.