Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p paralogs results in catalytically active succinate dehydrogenase isoenzymes

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.     

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q(P)) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the Q(P) site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.     

Cloning and characterization of the iron-sulfur subunit gene of succinate dehydrogenase from Saccharomyces cerevisiae

We describe the cloning and characterization of the complete gene for the iron-sulfur protein subunit of succinate dehydrogenase (EC 1.3.99.1) from Saccharomyces cerevisiae. The promoter and coding sequence have been cloned into an Escherichia coli-yeast shuttle vector. The cloned gene complements the defect in a succinate dehydrogenase-deficient yeast mutant isolated by us, and gene expression is fully responsive to induction by glucose deprivation, indicating that the promoter is intact.     

Ubiquinone-binding site mutations in the Saccharomyces cerevisiae succinate dehydrogenase generate superoxide and lead to the accumulation of succinate

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma. The mechanisms linking SDH mutations to oncogenesis are still unclear. In this work, we used the yeast SDH to investigate the molecular and catalytic effects of tumorigenic or related mutations. We mutated Arg(47) of the Sdh3p subunit to Cys, Glu, and Lys and Asp(88) of the Sdh4p subunit to Asn, Glu, and Lys. Both Arg(47) and Asp(88) are conserved residues, and Arg(47) is a known site of cancer causing mutations in humans. All of the mutants examined have reduced ubiquinone reductase activities. The SDH3 R47K, SDH4 D88E, and SDH4 D88N mutants are sensitive to hyperoxia and paraquat and have elevated rates of superoxide production in vitro and in vivo.We also observed the accumulation and secretion of succinate. Succinate can inhibit prolyl hydroxylase enzymes, which initiate a proliferative response through the activation of hypoxia-inducible factor 1alpha. We suggest that SDH mutations can promote tumor formation by contributing to both reactive oxygen species production and to a proliferative response normally induced by hypoxia via the accumulation of succinate.     

Isolation and characterization of a Saccharomyces cerevisiae mutant disrupted for the succinate dehydrogenase flavoprotein subunit

A partial genomic clone of the flavoprotein subunit of the mitochondrial enzyme, succinate dehydrogenase (EC 1.3.99.1) from Saccharomyces cerevisiae has been isolated. The partial clone was used to construct, by targeted gene disruption, a yeast mutant with a defective flavoprotein subunit gene. Submitochondrial membranes from the mutant are defective in activities requiring a functional succinate dehydrogenase but not in other respiratory chain activities. In addition, the mutant contains significantly lower levels of covalently attached flavin adenine dinucleotide cofactor than does the wild type. Disruption of the flavoprotein subunit gene results in the simultaneous loss of both the iron-sulfur and the flavoprotein subunits from mitochondrial membranes.     

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q_P) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the Q_P site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.     

The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase

Sirohaem is a tetrapyrrole-derived prosthetic group that is required for the essential assimilation of sulfur and nitrogen into all living systems as part of the sulfite and nitrite reductase systems. The final two steps in the biosynthesis of sirohaem involve a beta-NAD(+)-dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield sirohaem. In Saccharomyces cerevisiae, Met8p is a bifunctional enzyme that carries out both of these reactions. Here, we report the 2.2 A resolution crystal structure of Met8p, which adopts a novel fold that bears no resemblance to the previously determined structures of cobalt- or ferro-chelatases. Analysis of mutant proteins suggests that both catalytic activities share a single active site, and that Asp141 plays an essential role in both dehydrogenase and chelatase processes.     

The quaternary structure of the Saccharomyces cerevisiae succinate dehydrogenase. Homology modeling, cofactor docking, and molecular dynamics simulation studies

Succinate dehydrogenases and fumarate reductases are complex mitochondrial or bacterial respiratory chain proteins with remarkably similar structures and functions. Succinate dehydrogenase oxidizes succinate and reduces ubiquinone using a flavin adenine dinucleotide cofactor and iron-sulfur clusters to transport electrons. A model of the quaternary structure of the tetrameric Saccharomyces cerevisiae succinate dehydrogenase was constructed based on the crystal structures of the Escherichia coli succinate dehydrogenase, the E. coli fumarate reductase, and the Wolinella succinogenes fumarate reductase. One FAD and three iron-sulfur clusters were docked into the Sdh1p and Sdh2p catalytic dimer. One b-type heme and two ubiquinone or inhibitor analog molecules were docked into the Sdh3p and Sdh4p membrane dimer. The model is consistent with numerous experimental observations. The calculated free energies of inhibitor binding are in excellent agreement with the experimentally determined inhibitory constants. Functionally important residues identified by mutagenesis of the SDH3 and SDH4 genes are located near the two proposed quinone-binding sites, which are separated by the heme. The proximal quinone-binding site, located nearest the catalytic dimer, has a considerably more polar environment than the distal site. Alternative low energy conformations of the membrane subunits were explored in a molecular dynamics simulation of the dimer embedded in a phospholipid bilayer. The simulation offers insight into why Sdh4p Cys-78 may be serving as the second axial ligand for the heme instead of a histidine residue. We discuss the possible roles of heme and of the two quinone-binding sites in electron transport.     

Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain

The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S. cerevisiae strains. The osmotic water permeability coefficient (Pf) of X. laevis oocytes expressing the gene cloned from the Sigma1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DeltaCp), mediated an approximately 3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in Sigma1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.     

She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast,Saccharomyces cerevisiae

She4p/Dim1p, a member of the UNC-45/CRO1/She4p (UCS) domain-containing protein family, is required for endocytosis, polarization of actin cytoskeleton, and polarization of ASH1 mRNA in Saccharomyces cerevisiae. We show herein that She4p/Dim1p is involved in endocytosis and actin polarization through interactions with the type I myosins Myo3p and Myo5p. Two-hybrid and biochemical experiments showed that She4p/Dim1p interacts with the motor domain of Myo3/5p through its UCS domain. She4p/Dim1p was required for Myo5p localization to cortical patch-like structures. Using random mutagenesis of the motor region of MYO5, we identified four independent dominant point mutations that suppress the temperature-sensitive growth phenotype of the she4/dim1 null mutant. All of the amino acid substitutions caused by these mutations, V164I, N168I, N209S, and K377M, could suppress the defects of endocytosis and actin polarization of the she4/dim1 mutant as well. She4p/Dim1p also showed two-hybrid interactions with the motor domain of a type II myosin Myo1p and type V myosins Myo2p and Myo4p, and was required for proper localization of Myo4p, which regulates polarization of ASH1 mRNA. Our results suggest that She4p/Dim1p is required for structural integrity or regulation of the motor domain of unconventional myosins.     

Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae

The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. Recent work has identified Ccr4p and Pop2p as components of the major cytoplasmic deadenylase in yeast. We now demonstrate that CCR4 encodes the catalytic subunit of the deadenylase and that Pop2p is dispensable for catalysis. In addition, we demonstrate that at least some of the Ccr4p/Pop2p-associated Not proteins are cytoplasmic, and lesions in some of the NOT genes can lead to defects in mRNA deadenylation rates. The Ccr4p deadenylase is inhibited in vitro by addition of the poly(A) binding protein (Pab1p), suggesting that dissociation of Pab1p from the poly(A) tail may be rate limiting for deadenylation in vivo. In addition, the rapid deadenylation of the COX17 mRNA, which is controlled by a member of the Pumilio family of deadenylation activators Puf3p, requires an active Ccr4p/Pop2p/Not deadenylase. These results define the Ccr4p/Pop2p/Not complex as the cytoplasmic deadenylase in yeast and identify positive and negative regulators of this enzyme complex.     

The C-terminus of the succinate dehydrogenase IP peptide of Saccharomyces cerevisiae is significant for assembly of complex II.

Site-directed mutagenesis was used to introduce mutations into the gene for the iron protein (IP) of succinate dehydrogenase (SDH) of Saccharomyces cerevisiae. Specifically, three mutations were examined which caused the synthesis of truncated IP peptides missing four, seven, or 17 amino acids from the C-terminus, respectively. The deletion of seven or more amino acids includes the loss of two lysine residues, which appear to have been highly conserved in evolution. While the deletion of four amino acids had no effect on the assembly of complex II and on its activity, the deletion including the two lysines abolished SDS activity completely and led to the failure of the imported IP peptide to be incorporated into a stable complex II or SDH complex. Replacement of one of the lysines by threonine had no effect, but replacement of both by threonine affected the specific activity of complex II but not its assembly and stability.     

The Sla2p talin domain plays a role in endocytosis in Saccharomyces cerevisiae

Clathrin-binding adaptors play critical roles for endocytosis in multicellular organisms, but their roles in budding yeast have remained unclear. To address this question, we created a quadruple mutant yeast strain lacking the genes encoding the candidate clathrin adaptors Yap1801p, Yap1802p, and Ent2p and containing a truncated version of Ent1p, Ent1DeltaCBMp, missing its clathrin-binding motif. This strain was viable and competent for endocytosis, suggesting the existence of other redundant adaptor-like factors. To identify these factors, we mutagenized the quadruple clathrin adaptor mutant strain and selected cells that were viable in the presence of full-length Ent1p, but inviable with only Ent1DeltaCBMp; these strains were named Rcb (requires clathrin binding). One mutant strain, rcb432, contained a mutation in SLA2 that resulted in lower levels of a truncated protein lacking the F-actin binding talin homology domain. Analyses of this sla2 mutant showed that the talin homology domain is required for endocytosis at elevated temperature, that SLA2 exhibits genetic interactions with both ENT1 and ENT2, and that the clathrin adaptors and Sla2p together regulate the actin cytoskeleton and revealed conditions under which Yap1801p and Yap1802p contribute to viability. Together, our data support the view that Sla2p is an adaptor that links actin to clathrin and endocytosis.     

Gpi19, the Saccharomyces cerevisiae homologue of mammalian PIG-P, is a subunit of the initial enzyme for glycosylphosphatidylinositol anchor biosynthesis

Glycosylphosphatidylinositols (GPIs) are attached to the C termini of some glycosylated secretory proteins, serving as membrane anchors for many of those on the cell surface. Biosynthesis of GPIs is initiated by the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol. This reaction is carried out at the endoplasmic reticulum (ER) by an enzyme complex called GPI-N-acetylglucosaminyltransferase (GPI-GlcNAc transferase). The human enzyme has six known subunits, at least four of which, GPI1, PIG-A, PIG-C, and PIG-H, have functional homologs in the budding yeast Saccharomyces cerevisiae. The uncharacterized yeast gene YDR437w encodes a protein with some sequence similarity to human PIG-P, a fifth subunit of the GPI-GlcNAc transferase. Here we show that Ydr437w is a small but essential subunit of the yeast GPI-GlcNAc transferase, and we designate its gene GPI19. Similar to other mutants in the yeast enzyme, temperature-sensitive gpi19 mutants display cell wall defects and hyperactive Ras phenotypes. The Gpi19 protein associates with the yeast GPI-GlcNAc transferase in vivo, as judged by coimmuneprecipitation with the Gpi2 subunit. Moreover, conditional gpi19 mutants are defective for GPI-GlcNAc transferase activity in vitro. Finally, we present evidence for the topology of Gpi19 within the ER membrane.