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T H Stevens

Publications and source records attributed to T H Stevens.

88 records · Page 5Linked to original sources
PubMed1989

Protein sorting in yeast: the role of the vacuolar proton-translocating ATPase.

We are investigating the physiological roles of organelle acidification in yeast by two different approaches. First, we have identified two mutants which are defective in acidification of the yeast lysosome-like vacuole from among a collection of mutants which mis-sort soluble vacuolar proteins to the cell surface. These mutants have been helpful in identifying other cellular functions linked to acidification, such as the activation of vacuolar zymogens. We have complemented this classical genetic approach to acidification with direct biochemical and reverse genetic studies on the yeast vacuolar proton-translocating ATPase (H+-ATPase), the enzyme responsible for vacuolar network acidification. Our biochemical characterization of this enzyme indicates that it is a multisubunit complex with many structural similarities to other vacuolar H+-ATPases. Like the other vacuolar H+-ATPases characterized, it also shares some structural features with the F1F0-type ATPases of mitochondria, chloroplasts, and Escherichia coli. We are currently cloning the genes for the subunits of the yeast vacuolar H+-ATPase. Mutagenesis of the cloned genes will allow us to determine the phenotype of yeast cells expressing a vacuolar H+-ATPase altered in well controlled ways. We are also beginning to investigate how the subunits of the vacuolar H+-ATPase are assembled into the enzyme complex and targeted to their proper cellular location.

Biological Transport↗
PubMed1987

Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide.

We have isolated cis-acting mutations in the gene encoding the yeast vacuolar protein carboxypeptidase Y (CPY) that result in missorting and aberrant secretion of up to 95% of newly synthesized CPY. The CPY polypeptides synthesized by these mutants use the late secretory pathway to exit the cell, since the late-acting sec1 mutation prevents their secretion. The mutant versions of CPY are secreted as the proCPY zymogen and are enzymatically activatable in vivo and in vitro. All the mutations, including small deletions and an amino acid substitution, map to the amino-terminal propeptide region and define a discrete yeast vacuolar localization domain whose integrity is required for efficient sorting of the CPY zymogen. Thus, the N-terminal propeptide of CPY carries out at least three functions: it mediates translocation across the endoplasmic reticulum, renders the enzyme inactive during transit, and targets the molecule to the vacuole.

Amino Acid Sequence↗
PubMed1987

Yeast carboxypeptidase Y can be translocated and glycosylated without its amino-terminal signal sequence.

We have constructed a series of mutations in the signal sequence of the yeast vacuolar protein carboxypeptidase Y (CPY), and have used pulse-chase radiolabeling and immunoprecipitation to examine the in vivo effects of these mutations on the entry of the mutant CPY proteins into the secretory pathway. We find that introduction of a negatively charged residue, aspartate, into the hydrophobic core of the signal sequence has no apparent effect on signal sequence function. In contrast, internal in-frame deletions within the signal sequence cause CPY to be synthesized as unglycosylated precursors. These are slowly and inefficiently converted to glycosylated precursors that are indistinguishable from the glycosylated forms produced from the wild-type gene. These precursors are converted to active CPY in a PEP4-dependent manner, indicating that they are correctly localized to the vacuole. Surprisingly, a deletion mutation that removes the entire CPY signal sequence has a similar effect: unglycosylated precursor accumulates in cells carrying this mutant gene, and greater than 10% of it is posttranslationally glycosylated. Thus, the amino-terminal signal sequence of CPY, while important for translocation efficiency, is not absolutely required for the translocation of this protein.

Amino Acid Sequence↗
PubMed1986

Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway.

We have devised a genetic selection for mutant yeast cells that fail to properly deliver the vacuolar glycoprotein CPY to the lysosome-like vacuole. This has allowed us to identify mutations in eight VPL complementation groups that result in aberrant secretion of up to approximately 90% of the immunoreactive CPY. Other soluble vacuolar proteins are also affected by each vpl mutation, demonstrating that a sorting system for multiple vacuolar proteins exists in yeast. Mislocalized CPY apparently traverses late stages of the secretory pathway, since a vesicle-accumulating sec1 mutation prevents secretion of this protein. Despite the presence of abnormal membrane-enclosed organelles in some of the vpl mutants, maturation and secretion of invertase are not substantially perturbed. Thus vpl mutations define a new class of genes that encode products required for sorting of newly synthesized vacuolar proteins from secretory proteins during their transit through the yeast secretory pathway.

Aspartic Acid Endopeptidases↗
PubMed1986

Overproduction-induced mislocalization of a yeast vacuolar protein allows isolation of its structural gene.

Using an immunological screening procedure that allows the detection of yeast cells aberrantly secreting vacuolar proteins, we have isolated a cloned DNA fragment containing the structural gene for the vacuolar enzyme proteinase A (PrA; EC 3.4.23.6). A large portion of PrA is misdirected to the cell surface in cells harboring the PrA structural gene on a multicopy plasmid. This mislocalized PrA traverses the late stages of the secretory pathway and differs slightly in apparent molecular weight from the vacuolar form. A deletion in the genomic copy of the PrA structural gene eliminates immunoreactive PrA as well as the enzymatic activities of at least three other vacuolar hydrolases. In the case of the vacuolar enzyme carboxypeptidase Y (EC 3.4.16.1), the lack of activity is due to the absence of proteolytic activation of the zymogen. Thus, PrA may be required for in vivo processing of a number of yeast vacuolar hydrolases.

Aspartic Acid Endopeptidases↗
PubMed1986

Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y.

The structural gene for yeast vacuolar carboxypeptidase Y (PRC1) has been cloned by complementation of the prc1-1 mutation. As much as an eightfold elevation in the level of carboxypeptidase Y (CPY) results when a multiple-copy plasmid containing the PRC1 gene is introduced into yeast. Unlike the situation with a single copy of PRC1 in which newly synthesized CPY is efficiently localized to the vacuole, plasmid-directed overproduction results in secretion of greater than 50% of the protein as the precursor form. Secretion is blocked in a mutant that is defective at a late stage in the transport of periplasmic proteins. Unlike normal cell surface glycoproteins, secreted CPY precursor acquires no additional oligosaccharide modifications beyond those that accompany normal transport to the vacuole. In the periplasm, the CPY precursor is proteolytically activated to an enzymatically active form by an enzyme that is unrelated to the vacuolar processing enzyme. These findings suggest that proper sorting and transport of CPY is saturable. This may reflect limiting amounts of a CPY-sorting receptor, or of CPY-modifying machinery that is essential for recognition by such a receptor.

Carboxypeptidases↗
PubMed1986

PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors.

The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J. H. Rothman, C. P. Hunter, L. A. Valls, and T. H. Stevens, Proc. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.

Amino Acid Sequence↗
PubMed1983

Nuclear magnetic resonance studies of formyltetrahydrofolate synthetase interactions with formate and methylammonium ion.

Using nuclear magnetic resonance techniques, we have measured the internuclear distances separating the nucleotide-bound metal from the carbon and hydrogen nuclei of formate as well as the carbon of methylammonium cation when bound to formyltetrahydrofolate synthetase. Measurements were made of the paramagnetic effect on the spin-lattice relaxation rates (1/T1) of 13C and 1H nuclei arising from the replacement of Mg2+ with Mn2+, which binds to the enzyme in the form of a metal-nucleotide complex. Distances from Mn2+ to the formate carbon and proton were found to be 6.3 and 7.4 A, respectively, in the E . ATP . Mn2+ . formate complex and 6.0 and 7.1 A, respectively, in the E . ADP . Mn2+ . formate complex. When tetrahydrofolate was added to the latter complex, the exchange of formate was greatly reduced and became rate limiting for relaxation. These results are consistent with substantial conformational effects produced by the binding of the cofactor. The distance from Mn2+ to the methylammonium carbon in the E . ADP . Mn2+ . CH3NH+3, E . ADP . Mn2+ . formate . CH3NH3+, and E . ADP . Mn2+ . tetrahydrofolate . CH3NH3+ complexes was estimated to be in the range of 7.4-12 A. However, in the E . ADP . Mn2+ formate . tetrahydrofolate . CH3NH3+ complex, the data suggest that exchange of cation contributes significantly to relaxation. These results, combined with other known features of the enzyme, suggest that there may be a monovalent cation site within the active site of the enzyme.

Clostridium↗
PubMed1982

The nature of CuA in cytochrome c oxidase.

The isolation and purification of yeast cytochrome c oxidase is described. Characterization of the purified protein indicates that it is spectroscopically identical with cytochrome c oxidase isolated from beef heart. Preparations of isotopically substituted yeast cytochrome c oxidase are obtained incorporating [1,3-15N2]histidine or [beta,beta-2H2]cysteine. Electron paramagnetic resonance and electron nuclear double resonance spectra of the isotopically substituted proteins identify unambiguously at least 1 cysteine and 1 histidine as ligands to CuA and suggest that substantial spin density is delocalized onto a cysteine sulfur in the oxidized protein to render the site Cu(I)--S.

Copper↗
PubMed1981

Conformations of oxidized cytochrome c oxidase.

Oxidized cytochrome c oxidase is shown to exist in three conformations in addition to the transient "g5" conformation previously reported [Shaw, R. W., Hansen, R. E., & Beinert, H. (1978) J. Biol. Chem. 253, 6637-6640]. The "resting" and "g12" conformations are distinguished by an NO-induced cytochrome a3 electron paramagnetic resonance (EPR) signal and an EPR signal at g' = 12, respectively. The "oxygenated" conformatin exhibits an unusual EPR signal in the presence of fluoride and is identical with the "oxygenated" state first discovered by Okunuki et al. [Okunuki, K., Hagihora, B., Sekuzu, I., & Horio, T. (1958) Proc. Int. Symp. Enzyme Chem., Tokyo, Kyoto, 264]. It is proposed that when the reduced enzyme is reoxidized by dioxygen, the oxidized enzyme first relaxes from the "g5" into the "oxygenated" conformation after which a percentage of the molecules slowly relax into the "g12" conformation. The "resting" conformation is not formed when the enzyme is reoxidized. On the basis of the EPR observations, it is proposed that these various conformations of the oxidized enzyme differ in the structure of the cytochrome a3--Cua3 site. Structures for the cytochrome a3--Cua3 site are proposed for each conformation, and a mechanism by which these conformations undergo interconversion among themselves is described.

Animals↗
PubMed1980

Reactions of nitric oxide with cytochrome c oxidase.

The reactions of nitric oxide (NO) with both oxidized and reduced cytochrome c oxidase are reported. NMR and mass spectroscopy were utilized to determine the products of the reactions; EPR and optical spectroscopy were employed to determine the states of the enzyme produced in each of these reactions. It was found that the enzyme catalyzes the consecutive oxidation and reduction of NO. A different cycle was observed when NO was added to the reduced enzyme, to the oxidized enzyme, or to the oxidized enzyme in the presence of azide. It was possible to observe the state of the enzyme at several points in each of these three cycles by varying the concentration of NO. The reactions of NO all involved a one- or two-electron redox step and could be accounted for by the involvement of only cytochrome a3 and Cua3. On the basis of these results, a mechanism for the reduction of dioxygen by the enzyme is proposed in which cytochrome a3 functions to anchor dioxygen and intermediates while remaining in the ferrous state, whereas Cua3 functions to accept electroins from cytochrome a/Cua and transfer them to dioxygen.

Binding Sites↗
PubMed1980

Manganous ion binding to tubulin.

The assembly of pure tubulin into microtubules is induced by Mn(II), and the polymerized product displays the characteristic sensitivity of normal microtubules to low temperature. The interaction of Mn(II) with tubulin purified by phosphocellulose chromatography to remove microtubule-associated proteins was investigated using electron paramagnetic resonance and atomic absorption spectroscopy. Mn(II) interacts with tubulin at about one high affinity site with an apparent dissociation constant (KD) of 1.6 +/- 0.3 microM and at 8 +/- 2 low affinity (KD = 0.38 +/- 0.18 mM) sites. The binding of Mn(II) to tubulin at the high affinity site was accompanied by release of tubulin-bound Mg(II) from the protein. Thus, Mn(II) substitutes for Mg(II) at the high affinity site and in promoting the assembly of tubulin. Mg(II), Co(II), and Zn(II) can displace Mn(II) from manganese-tubulin, but Ca(II) cannot. The electron paramagnetic resonance spectrum at 9.1 GHz of Mn(II) bound to the high affinity divalent cation site of tubulin is characterized by broadened hyperfine structure and the absence of solid state spectral features, suggesting that the bulk solvent is accessible to the bound metal ion.

Animals↗
PubMed1979

Structure of cytochrome a3-Cua3 couple in cytochrome c oxidase as revealed by nitric oxide binding studies.

The addition of NO to oxidized cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high-spin heme electron paramagnetic resonance (EPR) signal due to cytochrome a3. This suggests that NO coordinates to Cu+2a3 and breaks the antiferromagnetic couple by forming a cytochrome a+33-Cu+2a3-NO complex. The intensity of the high-spin cytochrome a3 signal depends on the method of preparation of the enzyme and maximally accounts for 58% of one heme. The effect of N-3 on the cytochrome a+33-Cu+2a3-NO complex is to reduce cytochrome a3 to the ferrous state, and this is followed by formation of a new complex that exhibits EPR signals characteristic of a triplet species. On the basis of optical and EPR results, a NO bridge between cytochrome a+23 and Cu+2a3 is proposed--i.e., cytochrome a+23-NO-Cu+2a3. The half-field transition observed at g = 4.34 in the EPR spectrum of this triplet species exhibits resolved copper hyperfine splittings with [A+2] = 0.020 cm-1, indicating that the Cu+2a3 in the cytochrome a+23-NO-Cu+2a3 complex is similar to a type 2 copper site.

Animals↗