The copper metallochaperone Cox17 is proposed to shuttle Cu(I) ions to the mitochondrion for the assembly of cytochrome c oxidase. The Cu(I) ions are liganded by cysteinyl thiolates. Mutational analysis on the yeast Cox17 reveals three of the seven cysteinyl residues to be critical for Cox17 function, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines results in a nonfunctional cytochrome oxidase complex. Cells harboring such a mutation fail to grow on nonfermentable carbon sources and have no cytochrome c oxidase activity in isolated mitochondria. Wild-type Cox17 purified as untagged protein binds three Cu(I) ions/molecule. Mutant proteins lacking only one of these critical Cys residues retain the ability to bind three Cu(I) ions and are imported within the mitochondria. In contrast, Cox17 molecules with a double Cys 3 Ser mutation exhibit no Cu(I) binding but are still localized to the mitochondria. Thus, mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17. COX17 was originally cloned by virtue of complementation of a mutant containing a nonfunctional Cys 3 Tyr substitution at codon 57. The mutant C57Y Cox17 fails to accumulate within the mitochondria but retains the ability to bind three Cu(I) ions. A C57S Cox17 variant is functional, and a quadruple Cox17 mutant with C16S/C36S/C47S/C57S substitutions binds three Cu(I) ions. Thus, only three cysteinyl residues are important for the ligation of three Cu(I) ions. A novel mode of Cu(I) binding is predicted.

A current hypothesis explaining the toxicity of superoxide anion in vivo is that it oxidizes exposed [4Fe-4S] clusters in certain vulnerable enzymes causing release of iron and enzyme inactivation. The resulting increased levels of "free iron" catalyze deleterious oxidative reactions in the cell. In this study, we used low temperature Fe(III) electron paramagnetic resonance (EPR) spectroscopy to monitor iron status in whole cells of the unicellular eukaryote, Saccharomyces cerevisiae. The experimental protocol involved treatment of the cells with desferrioxamine, a cell-permeant, Fe(III)-specific chelator, to promote oxidation of all of the "free iron" to the Fe(III) state wherein it is EPR-detectable. Using this method, a small amount of EPR-detectable iron was detected in the wild-type strain, whereas significantly elevated levels were found in strains lacking CuZn-superoxide dismutase (CuZn-SOD) (sod1⌬), Mn-SOD (sod2⌬), or both SODs, throughout their growth but particularly in stationary phase. The accumulation was suppressed by expression of wild-type human CuZn-SOD (in the sod1⌬ mutant), by pmr1, a genetic suppressor of the sod⌬ mutant phenotype (in the sod1⌬sod2⌬ double knockout strain), and by anaerobic growth. In wild-type cells, an increase in the EPR-detectable iron pool could be induced by treatment with paraquat, a redox-cycling drug that generates superoxide. Cells that were not pretreated with desferrioxamine had Fe(III) EPR signals that were equally as strong as those from treated cells, indicating that "free iron" accumulated in the ferric form in our strains in vivo. Our results indicate a relationship between superoxide stress and iron handling and support the above hypothesis for superoxide-related oxidative damage.Superoxide dismutases are antioxidant enzymes that disproportionate superoxide (O 2 . ) (1) to hydrogen peroxide (H 2 O 2 ) and dioxygen (2-5). This class of enzyme is found in almost all aerobic organisms (6). Eukaryotes, including Saccharomyces cerevisiae, contain Mn-SOD 1 (product of the SOD2 gene) in the matrix of the mitochondria, and CuZn-SOD (product of the SOD1 gene) elsewhere in the cell, most notably in the cytoplasm, nucleus and lysosomes. Yeast cells lacking either SOD gene are viable but compromised in various ways. Yeast sod1⌬ mutant strains grow poorly in air, are very sensitive to redoxcycling drugs such as paraquat or menadione, die quickly in stationary phase, and exhibit lysine and methionine auxotrophies when grown aerobically. sod2⌬ mutants are oxygen-sensitive and, when required to respire, grow poorly and are particularly sensitive to paraquat. The double sod1⌬sod2⌬ mutant is more severely compromised, exhibiting essentially a summation of the single mutant phenotypes (7-9).We have been searching for explanations for the severity of the sod1 mutant phenotype in yeast, and thus for the toxicity of superoxide in vivo. Superoxide is more selective in its chemical reactions than most other reactive oxygen species and thus less likely to exert its toxicity throug...

The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change in response to zinc, suggesting that zinc regulates DNA binding and/or activation domain function. To understand how Zap1 responds to zinc, we performed a functional dissection of the protein. Zap1 contains two activation domains. DNA-binding activity is conferred by ®ve C-terminal C 2 H 2 zinc ®ngers and each ®nger is required for highaf®nity DNA binding. The zinc-responsive domain of Zap1 also maps to the C-terminal zinc ®ngers. Furthermore, mutations that disrupt some of these ®ngers cause constitutive activity of a bifunctional Gal4 DNA-binding domain±Zap1 fusion protein.These results demonstrate a novel function of Zap1 zinc ®ngers in zinc sensing as well as DNA binding.

Protocol-driven management of NP patients was associated with better hospital survival and survival to S2P. Among protocol elements, gastrostomy usage was linked to both improved hospital survival and survival to S2P. Home surveillance was associated with increased survival to S2P.

It has been widely reported that the white rot basidiomycete Phanerochaete chrysosporium, unlike most other white rot fungi, does not produce laccase, an enzyme implicated in lignin biodegradation. Our results showed that P. chrysosporium BKM-F1767 produces extracellular laccase in a defined culture medium containing cellulose (10 g/liter) and either 2.4 or 24 mM ammonium tartrate. Laccase activity was demonstrated in the concentrated extracellular culture fluids of this organism as determined by a laccase plate assay as well as a spectrophotometric assay with ABTS [2,2-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] as the substrate. Laccase activity was observed even after addition of excess catalase to the extracellular culture fluid to destroy the endogenously produced hydrogen peroxide, indicating that the observed activity is not due to a peroxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by activity staining with ABTS revealed the presence of a laccase band with an estimated M r of 46,500.