Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms.

The human pathogen Mycobacterium tuberculosis requires a P-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM).

High-affinity iron and calcium transport pathways are involved in U(VI) uptake in the budding yeast Saccharomyces cerevisiae.

Uranium (U) is a naturally-occurring radionuclide that is toxic for all living organisms. To date, the mechanisms of U uptake are far from being understood. Here we provide a direct characterization of the transport machineries capable of transporting U, using the yeast Saccharomyces cerevisiae as a unicellular eukaryote model.

Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex.

The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli.

The role of cysteine and sulfide in the interplay between microbial Hg(ii) uptake and sulfur metabolism.

Biogenic thiols, such as cysteine, have been used to control the speciation of Hg(ii) in bacterial exposure experiments. However, the extracellular biodegradation of excess cysteine leads to the formation of Hg(ii)-sulfide species, convoluting the interpretation of Hg(ii) uptake results. Herein, we test the hypothesis that Hg(ii)-sulfide species formation is a critical step during bacterial Hg(ii) uptake in the presence of excess cysteine.

Biophysical and structural characterization of the putative nickel chaperone CooT from Carboxydothermus hydrogenoformans.

Carboxydothermus hydrogenoformans is a model microorganism for the study of [NiFe]-CODH, a key enzyme of carbon cycle in anaerobic microorganisms. The enzyme possesses a unique active site (C-cluster), constituted of a distorted [NiFeS] cubane linked to a mononuclear Fe(II) center. Both the biogenesis of the C-cluster and the activation of CODH by nickel insertion remain unclear.

Functional characterization of the Ca-ATPase SMA1 from .

is a parasite that causes bilharzia, a neglected tropical disease affecting hundreds of millions of people each year worldwide. In 2012, had been identified as the only invertebrate possessing two SERCA-type Ca-ATPases, SMA1 and SMA2. However, our analysis of recent genomic data shows that the presence of two SERCA pumps is rather frequent in parasitic flatworms.

Structural Insights into the Nucleotide-Binding Domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana.

Copper is a crucial ion in cells, but needs to be closely controlled due to its toxic potential and ability to catalyse the formation of radicals. In chloroplasts, an important step for the proper functioning of the photosynthetic electron transfer chain is the delivery of copper to plastocyanin in the thylakoid lumen. The main route for copper transport to the thylakoid lumen is driven by two PIB-type ATPases, Heavy Metal ATPase 6 (HMA6) and HMA8, located in the inner membrane of the chloroplast envelope and in the thylakoid membrane, respectively.

Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPases.

Copper is an essential transition metal for living organisms. In the plant model Arabidopsis thaliana, half of the copper content is localized in the chloroplast, and as a cofactor of plastocyanin, copper is essential for photosynthesis. Within the chloroplast, copper delivery to plastocyanin involves two transporters of the PIB-1-ATPases subfamily: HMA6 at the chloroplast envelope and HMA8 in the thylakoid membranes.

Membrane Protein Production in Lactococcus lactis for Functional Studies.

Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.

HMA6 and HMA8 are two chloroplast Cu+-ATPases with different enzymatic properties.

Copper (Cu) plays a key role in the photosynthetic process as cofactor of the plastocyanin (PC), an essential component of the chloroplast photosynthetic electron transfer chain. Encoded by the nuclear genome, PC is translocated in its apo-form into the chloroplast and the lumen of thylakoids where it is processed to its mature form and acquires Cu. In Arabidopsis, Cu delivery into the thylakoids involves two transporters of the PIB-1 ATPases family, heavy metal associated protein 6 (HMA6) located at the chloroplast envelope and HMA8 at the thylakoid membrane.

Inhibition of the ferric uptake regulator by peptides derived from anti-FUR peptide aptamers: coupled theoretical and experimental approaches.

The FUR protein (ferric uptake regulator) is an iron-dependent global transcriptional regulator. Specific to bacteria, FUR is an attractive antibacterial target since virulence is correlated to iron bioavailability. Recently, four anti-FUR peptide aptamers, composed of 13 amino acid variable loops inserted into a thioredoxinA scaffold, were identified, which were able to interact with Escherichia coli FUR (EcFUR), inhibit its binding to DNA and to decrease the virulence of pathogenic E.

Yeast-based production and purification of HIS-tagged human ATAD3A, A specific target of S100B.

ATAD3 is a mitochondrial integral inner membrane ATPase with unknown function. ATAD3 is absent in yeast and protozoan and present in all pluricellular eucaryotes where its expression is essential for development. To date, bacterial-based expression of full-length ATAD3 has been unsuccessful because of very high levels of endogenous degradation.

Amyloid fibrils formed by the programmed cell death regulator Bcl-xL.

The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis.

Biochemical characterization of AtHMA6/PAA1, a chloroplast envelope Cu(I)-ATPase.

Copper is an essential plant micronutrient playing key roles in cellular processes, among them photosynthesis. In Arabidopsis thaliana, copper delivery to chloroplasts, mainly studied by genetic approaches, is thought to involve two P(IB)-type ATPases: AtHMA1 and AtHMA6/PAA1. The lack of biochemical characterization of AtHMA1 and PAA1, and more generally of plant P(IB)-type ATPases, is due to the difficulty of getting high amounts of these membrane proteins in an active form, either from their native environment or after expression in heterologous systems.

Endoplasmic reticulum is a major target of cadmium toxicity in yeast.

Cadmium (Cd(2+)) is a very toxic metal that causes DNA damage, oxidative stress and apoptosis. Despite many studies, the cellular and molecular mechanisms underlying its high toxicity are not clearly understood. We show here that very low doses of Cd(2+) cause ER stress in Saccharomyces cerevisiae as evidenced by the induction of the unfolded protein response (UPR) and the splicing of HAC1 mRNA.

CadA, the Cd2+-ATPase from Listeria monocytogenes, can use Cd2+ as co-substrate.

CadA is a membrane protein of the P-type ATPase family which is the major determinant of the resistance to Cd2+ in Listeria monocytogenes. During its catalytic cycle, CadA undergoes auto-phosphorylation from ATP at Asp398, which allows Cd2+ translocation across the membrane. In the reverse mode, Asp398 is phosphorylated from Pi.

The cadmium transport sites of CadA, the Cd2+-ATPase from Listeria monocytogenes.

CadA, the Cd(2+)-ATPase from Listeria monocytogenes, belongs to the Zn(2+)/Cd(2+)/Pb(2+)-ATPase bacterial subfamily of P(1B)-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd(2+), CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd(2+) (Wu, C. C.

Structural basis for metal binding specificity: the N-terminal cadmium binding domain of the P1-type ATPase CadA.

In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a betaalphabetabetaalphabeta fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy.

A cloned prokaryotic Cd2+ P-type ATPase increases yeast sensitivity to Cd2+.

CadA, the P1-type ATPase involved in Listeria monocytogenes resistance to Cd(2+), was expressed in Saccharomyces cerevisiae and did just the opposite to what was expected, as it strikingly decreased the Cd(2+) tolerance of these cells. Yeast cells expressing the non-functional mutant Asp(398)Ala could grow on selective medium containing up to 100 microM Cd(2+), whereas those expressing the functional protein could not grow in the presence of 1 microM Cd(2+). The CadA-GFP fusion protein was localized in the endoplasmic reticulum membrane, suggesting that yeast hyper-sensitivity was due to Cd(2+) accumulation in the reticulum lumen.

A mutational study in the transmembrane domain of Ccc2p, the yeast Cu(I)-ATPase, shows different roles for each Cys-Pro-Cys cysteine.

Ccc2p is homologous to the human Menkes and Wilson copper ATPases and is herein studied as a model for human copper transport. Most studies to date have sought to understand how mutations in the human Menkes or Wilson genes impair copper homeostasis and induce disease. Here we analyze whether eight conserved amino acids of the transmembrane domain are important for copper transport.

Cd2+ and the N-terminal metal-binding domain protect the putative membranous CPC motif of the Cd2+-ATPase of Listeria monocytogenes.

CadA, the Cd(2+)-ATPase of Listeria monocytogenes, contains four cysteine residues: two in the CTNC (Cys-Thr-Asn-Cys) sequence in the cytoplasmic metal-binding domain (MBD), and two in the CPC (Cys-Pro-Cys) sequence in the membrane domain. Taking advantage of DeltaMBD, a truncated version of CadA that lacks the MBD but which still acts as a functional Cd(2+)-ATPase [Bal, Mintz, Guillain and Catty (2001) FEBS Lett. 506, 249-252], we analysed the role of the membrane cysteine residues (studied using DeltaMBD) separately from that of the cysteine residues of the MBD, which were studied using full-length CadA.

A possible regulatory role for the metal-binding domain of CadA, the Listeria monocytogenes Cd2+-ATPase.

Using the baculovirus/Sf9 expression system, we produced CadA and DeltaMBD, a metal-binding domain, truncated CadA. Both proteins had the expected properties of P-type ATPases: ATP-induced Cd2+ accumulation, Cd2+-sensitive ATP and Pi phosphorylation and ATPase activity. DeltaMBD displayed lower initial transport velocity as well as lower maximal ATPase activity than CadA.

Purification of heterologous sarcoplasmic reticulum Ca2+-ATPase Serca1a allowing phosphoenzyme and Ca2+-affinity measurements.

We describe here a protocol to prepare milligrams of active and stable heterologous sarcoplasmic reticulum Ca(2+)-ATPase (Serca1a). Serca1a was tagged with 6 histidines at its C-terminal end and overexpressed using the baculovirus-Sf9 system. In a first trial, Serca1a accounted for 24% of membrane proteins, 95% of which were inactive.

A transmembrane domain of the sulfonylurea receptor mediates activation of ATP-sensitive K(+) channels by K(+) channel openers.

ATP-sensitive K(+) (K(ATP)) channels are a complex of an ATP-binding cassette transporter, the sulfonylurea receptor (SUR), and an inward rectifier K(+) channel subunit, Kir6.2. The diverse pharmacological responsiveness of K(ATP) channels from various tissues are thought to arise from distinct SUR isoforms.