Copper-Dependent Trafficking of the Ctr4-Ctr5 Copper Transporting Complex

In Schizosaccharomyces pombe, copper uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Copper-induced differential subcellular localization may play a critical role with respect to fine tuning the number of Ctr4 and Ctr5 molecules at the cell surface.We have developed a bimolecular fluorescence complementation (BiFC) assay to analyze protein-protein interactions in vivo in S. pombe. The assay is based on the observation that N- and C-terminal subfragments of the Venus fluorescent protein can reconstitute a functional fluorophore only when they are brought into tight contact. Wild-type copies of the ctr4(+) and ctr5(+) genes were inserted downstream of and in-frame with the nonfluorescent C-terminal (VC) and N-terminal (VN) coding fragments of Venus, respectively. Co-expression of Ctr4-VC and Ctr5-VN fusion proteins allowed their detection at the plasma membrane of copper-limited cells. Similarly, cells co-expressing Ctr4-VN and Ctr4-VC in the presence of Ctr5-Myc(12) displayed a fluorescence signal at the plasma membrane. In contrast, Ctr5-VN and Ctr5-VC co-expressed in the presence of Ctr4-Flag(2) failed to be visualized at the plasma membrane, suggesting a requirement for a combination of two Ctr4 molecules with one Ctr5 molecule. We found that plasma membrane-located Ctr4-VC-Ctr5-VN fluorescent complexes were internalized when the cells were exposed to high levels of copper. The copper-induced internalization of Ctr4-VC-Ctr5-VN complexes was not dependent on de novo protein synthesis. When cells were transferred back from high to low copper levels, there was reappearance of the BiFC fluorescent signal at the plasma membrane.These findings reveal a copper-dependent internalization and recycling of the heteromeric Ctr4-Ctr5 complex as a function of copper availability

Zinc-Regulated DNA Binding of the Yeast Zap1 Zinc-Responsive Activator

The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1

Regulation of Alr1 Mg Transporter Activity by Intracellular Magnesium

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni2+, Mn2+, Zn2+ and Co2+. Using Ni2+ uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms

A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

The bisulfite genomic sequencing protocol is a widely used method for analyzing DNA methylation. It relies on the deamination of unmethylated cytosine residues to uracil; however, its high rates of DNA degradation and incomplete cytosine to uracil conversion often lead to failed experiments, uninformative results, and false positives. Here, we report the addition of a single-step multiple restriction enzyme digestion (MRED) designed to differentially digest polymerase chain reaction products amplified from unconverted DNA while leaving those of converted DNA intact. We show that for our model system, RARB2 P2 promoter, use of MRED increased informative sequencings ninefold, and MRED did not alter the clonal representation in one fully methylated cell line, H-596, treated or not with 5-azadeoxycytidine, a methylation inhibitor. We believe that this method may easily be adapted for analyzing other genes and provide guidelines for selecting the most appropriate MRED restriction enzymes

Cytosolic Superoxide Dismutase (SOD1) Is Critical for Tolerating the Oxidative Stress of Zinc Deficiency in Yeast

Zinc deficiency causes oxidative stress in many organisms including the yeast Saccharomyces cerevisiae. Previous studies of this yeast indicated that the Tsa1 peroxiredoxin is required for optimal growth in low zinc because of its role in degrading H2O2. In this report, we assessed the importance of other antioxidant genes to zinc-limited growth. Our results indicated that the cytosolic superoxide dismutase Sod1 is also critical for growth under zinc-limiting conditions. We also found that Ccs1, the copper-delivering chaperone required for Sod1 activity is essential for optimal zinc-limited growth. To our knowledge, this is the first demonstration of the important roles these proteins play under this condition. It has been proposed previously that a loss of Sod1 activity due to inefficient metallation is one source of reactive oxygen species (ROS) under zinc-limiting conditions. Consistent with this hypothesis, we found that both the level and activity of Sod1 is diminished in zinc-deficient cells. However, under conditions in which Sod1 was overexpressed in zinc-limited cells and activity was restored, we observed no decrease in ROS levels. Thus, these data indicate that while Sod1 activity is critical for low zinc growth, diminished Sod1 activity is not a major source of the elevated ROS observed under these conditions

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals toxic effects observed.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes" (NORTE-07-0124-FEDER-000028), Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. Manuela D. Machado gratefully acknowledges the post-doctoral grant from FCT (SFRH/BPD/72816/2010). Vanessa A. Mesquita gratefully acknowledges the grant from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES). The authors also thank to Doctor Rosane Freitas Schwan to offer the yeast strain and to Doctor Helena M.V.M. Soares, from the Faculty of Engineering of Porto University, for the use of analytical facilities (AAS with flame atomization and AAS with electrothermal atomization)

Genome-Wide Functional Profiling Identifies Genes and Processes Important for Zinc-Limited Growth of Saccharomyces cerevisiae

Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. Mixed pools of ∼4,600 deletion mutants were inoculated into zinc-replete and zinc-limiting media. These cells were grown for several generations, and the prevalence of each mutant in the pool was then determined by microarray analysis. As a result, we identified more than 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the ICE2 gene in maintaining ER zinc homeostasis. Thus, functional profiling has provided many new insights into genes and processes that are needed for cells to thrive under the stress of zinc deficiency

Interaction of the Deubiquitinating Enzyme Ubp2 and the E3 Ligase Rsp5 Is Required for Transporter/Receptor Sorting in the Multivesicular Body Pathway

Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis

Endocytic regulation of alkali metal transport proteins in mammals, yeast and plants

The relative concentrations of ions and solutes inside cells are actively maintained by several classes of transport proteins, in many cases against their concentration gradient. These transport processes, which consume a large portion of cellular energy, must be constantly regulated. Many structurally distinct families of channels, carriers, and pumps have been characterized in considerable detail during the past decades and defects in the function of some of these proteins have been linked to a growing list of human diseases. The dynamic regulation of the transport proteins present at the cell surface is vital for both normal cellular function and for the successful adaptation to changing environments. The composition of proteins present at the cell surface is controlled on both the transcriptional and post-translational level. Post-translational regulation involves highly conserved mechanisms of phosphorylation- and ubiquitylation-dependent signal transduction routes used to modify the cohort of receptors and transport proteins present under any given circumstances. In this review, we will summarize what is currently known about one facet of this regulatory process: the endocytic regulation of alkali metal transport proteins. The physiological relevance, major contributors, parallels and missing pieces of the puzzle in mammals, yeast and plants will be discussed.This work was supported by grant BFU2011-30197-C03-03 from the Ministerio de Ciencia e Innovacion (Spain). V.L.-T. is supported by a fellowship from the Universidad Politecnica de Valencia. C. P. is supported by a fellowship from the Consejo Superior de Investigaciones Cientificas (Spain).Mulet Salort, JM.; Llopis Torregrosa, V.; Primo Planta, C.; Marques Romero, MC.; Yenush, L. (2013). 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