Michel Ghislain

The Arabidopsis Multistress Regulator TSPO Is a Heme Binding Membrane Protein and a Potential Scavenger of Porphyrins via an Autophagy-Dependent Degradation Mechanism

This study establishes that plant TSPO, an enigmatic stress-induced membrane protein, binds porphyrins, including heme in vitro and in vivo, and that heme binding regulates TSPO degradation through autophagy. This work indicates that TSPO expression may, at least in part, transiently protect plant cell from porphyrin-induced damages. TSPO, a stress-induced, posttranslationally regulated, early secretory pathway-localized plant cell membrane protein, belongs to the TspO/MBR family of regulatory proteins, which can bind porphyrins. This work finds that boosting tetrapyrrole biosynthesis enhanced TSPO degradation in Arabidopsis thaliana and that TSPO could bind heme in vitro and in vivo. This binding required the His residue at position 91 (H91), but not that at position 115 (H115). The H91A and double H91A/H115A substitutions stabilized TSPO and rendered the protein insensitive to heme-regulated degradation, suggesting that heme binding regulates At-TSPO degradation. TSPO degradation was inhibited in the autophagy-defective atg5 mutant and was sensitive to inhibitors of type III phosphoinositide 3-kinases, which regulate autophagy in eukaryotic cells. Mutation of the two Tyr residues in a putative ubiquitin-like ATG8 interacting motif of At-TSPO did not affect heme binding in vitro but stabilized the protein in vivo, suggesting that downregulation of At-TSPO requires an active autophagy pathway, in addition to heme. Abscisic acid–dependent TSPO induction was accompanied by an increase in unbound heme levels, and downregulation of TSPO coincided with the return to steady state levels of unbound heme, suggesting that a physiological consequence of active TSPO downregulation may be heme scavenging. In addition, overexpression of TSPO attenuated aminolevulinic acid–induced porphyria in plant cells. Taken together, these data support a role for TSPO in porphyrin binding and scavenging during stress in plants.

Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1.

Nicotinamidase (NAMase) from the budding yeast, Saccharomyces cerevisiae, was purified by Ni2+ affinity chromatography and gel filtration. N‐terminal microsequencing revealed sequence identity with a hypothetical polypeptide encoded by the yeast YGL037C open reading frame sharing 30% sequence identity with Escherichia coli pyrazinamidase/nicotinamidase. A yeast strain in which the NAMase gene, hereafter named PNC1, was deleted shows a decreased intracellular NAD+ concentration, consistent with the loss of NAMase activity in the null mutant. In wild‐type strains, NAMase activity is stimulated during the stationary phase of growth, by various hyperosmotic shocks or by ethanol treatment. Using a PPNC1::lacZ gene fusion, we have shown that this stimulation of NAMase activity results from increased levels of the protein and requires stress response elements in the 5′ non‐coding region of PNC1. These results suggest that NAMase helps yeast cells to adapt to various stress conditions and nutrient depletion, most likely via the activation of NAD‐dependent biological processes. Copyright

Control of 26S proteasome expression by transcription factors regulating multidrug resistance in Saccharomyces cerevisiae

In eukaryotic cells, intracellular proteolysis occurs mainly via the ubiquitin–proteasome system. Expression of the yeast proteasome is under the control of the transcription factor, Rpn4p (also known as Son1p/Ufd5p). We show here that the RPN4 gene promoter contains regulatory sequences that bind Pdr1p and Pdr3p, two homologous zinc finger‐containing transcription factors, which mediate multiple drug resistance through the expression of membrane transporter proteins. Mutations in the RPN4 Pdr1p/Pdr3p binding sites lead to decreased expression of the proteasome RPT6 gene and to defective ubiquitin‐mediated proteolysis. Pdr3p, but not Pdr1p, is required for normal levels of intracellular proteolysis, indicating that the two transcription factors have distinct functions in the control of RPN4 expression. The RPN4 promoter contains an additional sequence that binds Yap1p, a bZIP‐type transcription factor that plays an important role in the oxidative stress response and multidrug resistance. We also show that the Yap1p response element is important in the transactivation of RPN4 by Yap1p. In yeast cells lacking Pdr1p, ubiquitin‐Pro‐β‐galactosidase, a short‐lived protein used to assay proteasome activity, is stabilized by the loss of Yap1p. These data demonstrate that the ubiquitin–proteasome system is controlled by transcriptional regulators of multidrug resistance via RPN4 expression.

Phosphorylation of Yeast Plasma Membrane H+-ATPase by Casein Kinase I

The plasma membrane H+-ATPase of Saccharomyces cerevisiae is subject to phosphorylation by a casein kinase I activity in vitro. We show this casein kinase I activity to result from the combined function of YCK1 and YCK2, two highly similar and plasma membrane-associated casein kinase I homologues. First, H+-ATPase phosphorylation is severely impaired in the plasma membrane of YCK-deficient yeast strains. Furthermore, the wild-type level of the phosphoprotein is restored by the addition of purified mammalian casein kinase I to the mutant membranes. We used the H+-ATPase as well as a synthetic peptide substrate that contains a phosphorylation site for casein kinase I to compare kinase activity in membranes prepared from yeast cells grown in the presence or absence of glucose. The addition of glucose results in increased H+-ATPase activity which is associated with a decline in the phosphorylation level of the enzyme. Mutations in both YCK1 and YCK2 affect this regulation, suggesting that H+-ATPase activity is modulated by glucose via a combination of a “down-regulating” casein kinase I activity and another, yet uncharacterized, “up-regulating” kinase activity. Biochemical mapping of phosphorylated H+-ATPase identifies a major phosphopeptide that contains a consensus phosphorylation site (Ser-507) for casein kinase I. Site-directed mutagenesis of this consensus sequence indicates that Glu-504 is important for glucose-induced decrease in the apparent Km for ATP.

Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway.

Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathways ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, and His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, and Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first ∼700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated ∼70-residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with a Kd of ∼1 μm, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of cognate dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1 type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.

Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation.

The Cdc48/p97 AAA‐ATPase functions in membrane fusion and ubiquitin‐dependent protein degradation. Here, we show that, in yeast, Cdc48p interacts with three novel proteins, Cuil–3p, which contain a conserved ubiquitin‐related (UBX) domain. Cui2p and Cui3p are closely related, interact with each other, and are localized at the perinuclear membrane. Cdc48p binds directly the UBX domain of Cui3p in vitro. Multiple deletions of the CUI1, CUI2 and CUI3 genes confer deficiency in sporulation and degradation of model ubiquitin–protein fusions. The Cuil–3 proteins were also found to interact with Ufd3p, a WD repeat protein known to associate with Cdc48p. Together, these results indicate that the Cuil–3 proteins form complexes that are components of the ubiquitin–proteasome system. Copyright

Casein Kinase I-dependent Phosphorylation and Stability of the Yeast Multidrug Transporter Pdr5p

The pleiotropic drug resistance protein, Pdr5p, is an ATP-binding cassette transporter of the plasma membrane ofSaccharomyces cerevisiae. Overexpression of Pdr5p results in increased cell resistance to a variety of cytotoxic compounds, a phenotype reminiscent of the multiple drug resistance seen in tumor cells. Pdr5p and two other yeast ATP-binding cassette transporters, Snq2p and Yor1p, were found to be phosphorylated on serine residuesin vitro. Mutations in the plasma membrane-bound casein kinase I isoforms, Yck1p and Yck2p, abolished Pdr5p phosphorylation and modified the multiple drug resistance profile. We showed Pdr5p to be ubiquitylated when overexpressed. However, instability of Pdr5p was only seen in Yck1p- and Yck2p-deficient strains, in which it was degraded in the vacuole via a Pep4p-dependent mechanism. Our results suggest that casein kinase I activity is required for membrane trafficking of Pdr5p to the cell surface. In the absence of functional Yck1p and Yck2p, Pdr5p is transported to the vacuole for degradation.

Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance.

SERCA1a, the fast‐twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca2+‐ATPase, was expressed in yeast using the promoter of the plasma membrane H+‐ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca2+‐ATPase and the vacuole PMC1 Ca2+‐ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin‐dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intracellular compartments.

Schistosoma mansoni Ca2+-ATPase SMA2 restores viability to yeast Ca2+-ATPase-deficient strains and functions in calcineurin-mediated Ca2+ tolerance.

The sarco(endo)plasmic reticulum of animal cells contains an ATP-powered Ca2+ pump that belongs to the P-type family of membrane-bound cation-translocating enzymes. InSchistosoma mansoni, the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is encoded by the SMA1 andSMA2 genes. A full-length SMA2 cDNA clone was isolated, sequenced, and expressed into a yeast Ca2+-ATPase-deficient strain requiring plasmid-borne rabbit SERCA1a for viability. The S. mansoniCa2+-ATPase supports growth of mutant cells lacking SERCA1a, indicating functional expression in yeast and a role in calcium sequestration. Subcellular fractionation showed that the SMA2 ATPase is localized in yeast internal membranes. SMA2 expression was found to be associated with thapsigargin-sensitive, Ca2+-dependent ATPase activity. The activity increased 2-fold upon calcineurin inactivation, which correlates within vivo stimulated contribution of SMA2 in calcium tolerance. These results suggest that calcineurin controls calcium homeostasis by inhibiting Ca2+-ATPase activity in an internal compartment.

The lipid-translocating exporter family and membrane phospholipid homeostasis in yeast.

The fungal lipid-translocating exporter family consists of conserved membrane proteins, with six or seven transmembrane spans. Phylogenetic trees and conserved gene order relationships show that the common ancestor of five closely related hemiascomycetous yeast species contained the RSB1 and PUG1 paralogous genes. In Saccharomyces cerevisiae, Rsb1 functions as a transporter or translocase of sphingoid bases, whereas Pug1 facilitates the inducible transport of protoporphyrin IX and hemin. The budding yeast contains two other paralogs, Ylr046p, of unknown function, and Rta1p, overexpression of which confers resistance to an ergosterol biosynthesis inhibitor. Large-scale mRNA expression profiling has shown that transcription of PUG1, RTA1 and YLR046 is induced under hypoxic conditions. Ergosterol biosynthesis is impaired under low-oxygen conditions as a consequence of the decreased synthesis of heme and heme-containing proteins. These genes may encode transporters or sensors that facilitate the excretion of excessive or aberrant biosynthetic intermediates, either directly or indirectly. The expression of RSB1 and RTA1 is under the control of pleiotropic drug resistance transcription factors, suggesting that the encoded proteins may have additional roles in cell resistance to xenobiotics. This review summarizes current knowledge concerning the lipid-translocating exporter family and its potential functions, focusing on multidrug resistance and membrane phospholipid homeostasis.