Biomedical subjects

B Guiard

Publications and source records attributed to B Guiard.

At least 19 recordsLinked to original sources

PubMed2004

A new Hansenula polymorpha HAP4 homologue which contains only the N-terminal conserved domain of the protein is fully functional in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the HAP transcriptional complex is involved in the fermentation-respiration shift. This complex is composed of four subunits. Three subunits are necessary for DNA-binding, whereas the Hap4p subunit, glucose-repressed, contains the transcriptional activation domain. Hap4p is the key regulator of the complex activity in response to carbon sources in S. cerevisiae. To date, no HAP4 homologue has been identified, except in Kluyveromyces lactis. Examination of these two HAP4 sequences led to the identification of two very short conserved peptides also identified in other yeasts. In the yeast Hansenula polymorpha, two possible HAP4 homologues have been found. Their deduced amino acid sequences are similar to the ScHap4p and KlHap4p proteins only in the N-terminal 16-amino-acid basic motif. Since molecular genetic tools exist and complete genome sequence is known for this yeast, we expressed one of these putative HpHap4 proteins in S. cerevisiae and showed that this protein is able to restore the growth defect of the S. cerevisiae hap4-deleted strain. A set of experiments was performed to confirm the functional homology of this new gene with ScHAP4. The discovery of a Hap4-regulatory protein in H. polymorpha with only the N-terminal conserved domain of the S. cerevisiae protein indicates that this domain may play a crucial role during evolution.

CCAAT-Binding Factor↗

PubMed2001

Co-ordinate regulation of lactate metabolism genes in yeast: the role of the lactate permease gene JEN1.

In the yeast Saccharomyces cerevisiae, the first step in lactate metabolism is its transport across the plasma membrane, a proton symport process mediated by the product of the gene JEN1. Under aerobic conditions, the expression of JEN1 is regulated by the carbon source: the gene is repressed by glucose and induced by non-fermentable substrates. JEN1 expression is also controlled by oxygen availability, but is unaffected by the absence of haem biosynthesis. JEN1 is negatively regulated by the repressors Mig1p and Mig2p, and requires Cat8p for full derepression. In this report we demonstrate that, in addition to these regulators, the Hap2/3/4/5 complex interacts specifically with a CAAT-box element in the JEN1 promoter, and acts to derepress JEN1 expression. We also provide evidence for transcriptional stimulation of JEN1 by the protein kinase Snf1p. Data are presented which provide a better understanding of the molecular mechanisms implicated in the co-regulation of genes involved in the metabolism of lactate.

Aerobiosis↗

PubMed2001

The mitochondrial Hsp70-dependent import system actively unfolds preproteins and shortens the lag phase of translocation.

Unfolding is an essential process during translocation of preproteins into mitochondria; however, controversy exists as to whether mitochondria play an active role in unfolding. We have established an in vitro system with a kinetic saturation of the mitochondrial import machinery, yielding translocation rates comparable to in vivo import rates. Preproteins with short N-terminal segments in front of a folded domain show a characteristic delay of the onset of translocation (lag phase) although the maximal import rate is similar to that of longer preproteins. The lag phase is shortened by extending the N-terminal segment to improve the accessibility to matrix heat shock protein 70 and abolished by unfolding of the preprotein. A mutant mtHsp70 defective in binding to the inner membrane prolongs the lag phase and reduces the translocation activity. A direct comparison of the rate of spontaneous unfolding in solution with that during translocation demonstrates that unfolding by mitochondria is significantly faster, proving an active unfolding process. We conclude that access of mtHsp70 to N-terminal preprotein segments is critical for active unfolding and initiation of translocation.

Animals↗

PubMed2000

Tim23 links the inner and outer mitochondrial membranes.

Tim23, a key component of the mitochondrial preprotein translocase, is anchored in the inner membrane by its C-terminal domain and exposes an intermediate domain in the intermembrane space that functions as a presequence receptor. We show that the N-terminal domain of Tim23 is exposed on the surface of the outer membrane. The two-membrane-spanning topology of Tim23 is a novel characteristic in membrane biology. By the simultaneous integration into two membranes, Tim23 forms contacts between the outer and inner mitochondrial membranes. Tethering the inner membrane translocase to the outer membrane facilitates the transfer of precursor proteins from the TOM complex to the TIM23 complex and increases the efficiency of protein import.

Animals↗

PubMed2000

Heme-mediated transcriptional control in Kluyveromyces lactis.

Heme is of great importance in oxygen-dependent biological functions, since it serves as a prosthetic group for many proteins related to oxygen-binding, oxidative damage prevention and electron transport. It also regulates gene expression through the action of specific transcriptional regulatory factors. In this paper, we present an analysis of heme-dependent transcriptional regulation of several respiration-related genes in an aerobic respiratory yeast, Kluyveromyces lactis. We also report that the KlHEM13 gene, encoding the heme biosynthetic enzyme coproporphyrinogen oxidase, is under heme and oxygen transcriptional regulation, thereby controlling the synthesis of the effector, heme. KlHEM13 is induced during hypoxia, which represents the first report of a transcriptionally regulated gene with this behaviour in K. lactis.

Base Sequence↗

PubMed2000

Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface.

Two AAA proteases, each with its catalytic site at the opposite membrane surface, mediate the ATP-dependent degradation of mitochondrial inner membrane proteins. We demonstrate here that a model substrate polypeptide containing hydrophilic domains at both sides of the membrane can be completely degraded by either of the AAA proteases, if solvent-exposed domains are in an unfolded state. A short protein tail protruding from the membrane surface is sufficient to allow the proteolytic attack of an AAA protease that facilitates domain unfolding at the opposite side. Our results provide a rationale for the membrane arrangement of AAA proteases in mitochondria and demonstrate that degradation of membrane proteins by AAA proteases involves an active extraction of transmembrane segments and transport of solvent-exposed domains across the membrane.

Adenosine Triphosphatases↗

PubMed2000

Regulation of the CYB2 gene expression: transcriptional co-ordination by the Hap1p, Hap2/3/4/5p and Adr1p transcription factors.

Expression of the Saccharomyces cerevisiae nuclear gene CYB2 encoding the mitochondrial enzyme L-(+)-lactate-cytochrome c oxidoreductase (EC 1.2.2.3) is subject to several strict metabolic controls at the transcriptional level: repression due to glucose fermentation, derepression by ethanol, induction by lactate and inhibition under anaerobic conditions or in response to deficiency of haem biosynthesis. In this respect, the data obtained from the transcriptional analysis of the CYB2 gene contribute to a better understanding of the control of mitochondrial biogenesis. In this study, we show that Hap1p is the main transcriptional activator involved in the control of CYB2 transcription. We found that Hap1p activity, known to be oxygen dependent, is effected by DNA-protein interaction with two binding sites present in the CYB2 promoter. Control is moreover dependent on carbon sources. This regulation by the carbon substrates is subordinate to the activity of the complex Hap2/3/4/5p, which counteracts the negative effect of the URS1 element. Finally, our results suggest that the Adr1p transcriptional activator is also required in CYB2 transcription control. This work provides new data which allows a better understanding of the molecular mechanisms implicated in the co-regulation at the transcriptional level of the genes encoding proteins involved in various aspects of oxidative metabolism.

Animals↗

PubMed2000

Membrane potential-driven protein import into mitochondria. The sorting sequence of cytochrome b(2) modulates the deltapsi-dependence of translocation of the matrix-targeting sequence.

The transport of preproteins into or across the mitochondrial inner membrane requires the membrane potential Deltapsi across this membrane. Two roles of Deltapsi in the import of cleavable preproteins have been described: an electrophoretic effect on the positively charged matrix-targeting sequences and the activation of the translocase subunit Tim23. We report the unexpected finding that deletion of a segment within the sorting sequence of cytochrome b(2), which is located behind the matrix-targeting sequence, strongly influenced the Deltapsi-dependence of import. The differential Deltapsi-dependence was independent of the submitochondrial destination of the preprotein and was not attributable to the requirement for mitochondrial Hsp70 or Tim23. With a series of preprotein constructs, the net charge of the sorting sequence was altered, but the Deltapsi-dependence of import was not affected. These results suggested that the sorting sequence contributed to the import driving mechanism in a manner distinct from the two known roles of Deltapsi. Indeed, a charge-neutral amino acid exchange in the hydrophobic segment of the sorting sequence generated a preprotein with an even better import, i.e. one with lower Deltapsi-dependence than the wild-type preprotein. The sorting sequence functioned early in the import pathway since it strongly influenced the efficiency of translocation of the matrix-targeting sequence across the inner membrane. These results suggest a model whereby an electrophoretic effect of Deltapsi on the matrix-targeting sequence is complemented by an import-stimulating activity of the sorting sequence.

Amino Acid Sequence↗

PubMed1999

An internal targeting signal directing proteins into the mitochondrial intermembrane space.

Import of most nucleus-encoded preproteins into mitochondria is mediated by N-terminal presequences and requires a membrane potential and ATP hydrolysis. Little is known about the chemical nature and localization of other mitochondrial targeting signals or of the mechanisms by which they facilitate membrane passage. Mitochondrial heme lyases lack N-terminal targeting information. These proteins are localized in the intermembrane space and are essential for the covalent attachment of heme to c type cytochromes. For import of heme lyases, the translocase of the mitochondrial outer membrane complex is both necessary and sufficient. Here, we report the identification of the targeting signal of mitochondrial heme lyases in the third quarter of these proteins. The targeting sequence is highly conserved among all known heme lyases. Its chemical character is hydrophilic because of a large fraction of both positively and negatively charged amino acid residues. These features clearly distinguish this signal from classical presequences. When inserted into a cytosolic protein, the targeting sequence directs the fusion protein into the intermembrane space, even in the absence of a membrane potential or ATP hydrolysis. The heme lyase targeting sequence represents the first topogenic signal for energy-independent transport into the intermembrane space and harbors two types of information. It assures accurate recognition and translocation by the translocase of the mitochondrial outer membrane complex, and it is responsible for driving the import reaction by undergoing high-affinity interactions with components of the intermembrane space.

Amino Acid Sequence↗

PubMed1999

Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase.

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.

Biological Transport↗

PubMed1999

N-terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic "positive-inside" rule of membrane protein topology.

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N- and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for sec-independent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.

Biological Transport↗

PubMed1999

Regulation of the Saccharomyces cerevisiae DLD1 gene encoding the mitochondrial protein D-lactate ferricytochrome c oxidoreductase by HAP1 and HAP2/3/4/5.

Expression of the nuclear gene encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase (D-LCR) was investigated in Saccharomyces cerevisiae. This gene (DLD1) was found to be subject to several regulatory controls at the transcriptional level: synthesis of DLD1 mRNA is repressed by glucose, is derepressed in ethanol or lactate and is heme dependent. We therefore examined the role of the heme-dependent transcriptional activator Hap1p and the carbon source-dependent Hap2/3/4/5 complex. We found that the Hap2/3/4/5 complex and Hap1p have additive effects on the activation of DLD1 transcription: the Hap2/3/4/5 complex is necessary for DLD1 derepression following a shift from fermentable to non-fermentable carbon sources, while the Hap1p effect was independent of the carbon sources tested. An upstream region required for expression and regulation of the DLD1 gene was identified. Within this region the binding sites for both the Hap2/3/4/5 complex and Hap1p were defined by gel retardation experiments and site-directed mutagenesis. Comparison between sequences recognized by Hap1p in different promoters showed that the Hap1p binding site in the DLD1 promoter diverges from the consensus Hap1p binding site.

Base Sequence↗

PubMed1998

Unfolding of preproteins upon import into mitochondria.

Unfolding of preproteins and translocation across the mitochondrial membranes requires their interaction with mt-Hsp70 and Tim44 at the inner face of the inner membrane and ATP as an energy source. We measured the temperature dependence of the rates of unfolding and import into the matrix of two folded passenger domains, the tightly folded heme-binding domain (HBD) of cytochrome b2 and the loosely folded mouse dihydrofolate reductase (DHFR). Despite the stability of the HBD, its rates of thermal breathing were fast and the preprotein was imported rapidly at all temperatures. In contrast, rates of unfolding and import of DHFR were strongly temperature dependent and import was significantly slower than unfolding. In addition, import rates of DHFR were strongly dependent on the length of the presequence. We propose that the mitochondrial import motor does not exert a constant pulling force. Rather, mt-Hsp70 appears to release a translocating polypeptide chain such that the precursor can then slide back and refold on the surface of the mitochondria. Refolding competes with translocation, and passengers may undergo several rounds of unfolding and refolding prior to their import.

Adenosine Triphosphate↗

PubMed1998

The formation of respiratory chain complexes in mitochondria is under the proteolytic control of the m-AAA protease.

Yta10p (Afg3p) and Yta12p (Rcal1p), members of the conserved AAA family of ATPases, are subunits of the mitochondrial m-AAA protease, an inner membrane ATP-dependent metallopeptidase. Deletion of YTA10 or YTA12 impairs degradation of non-assembled inner membrane proteins and assembly of respiratory chain complexes. Mutations of the proteolytic sites in either YTA10 or YTA12 have been shown to inhibit proteolysis of membrane-integrated polypeptides but not the respiratory competence of the cells, suggesting additional activities of Yta10p and Yta12p. Here we demonstrate essential proteolytic functions of the m-AAA protease in the biogenesis of the respiratory chain. Cells harbouring proteolytically inactive forms of both Yta10p and Yta12p are respiratory deficient and exhibit a pleiotropic phenotype similar to Deltayta10 and Deltayta12 cells. They show deficiencies in expression of the intron-containing mitochondrial genes COX1 and COB. Splicing of COX1 and COB transcripts is impaired in mitochondria lacking m-AAA protease, whilst transcription and translation can proceed in the absence of Yta10p or Yta12p. The function of the m-AAA protease appears to be confined to introns encoding mRNA maturases. Our results reveal an overlapping substrate specificity of the subunits of the m-AAA protease and explain the impaired assembly of respiratory chain complexes by defects in expression of intron-containing genes in mitochondria lacking m-AAA protease.

Adenosine Triphosphatases↗

PubMed1998

Evidence of an overlap between the two half-sites of UAS1-B/CYC1--a new model for Cyp1p (Hap1p) DNA binding.

Cyp1p (Hap1p) is a yeast transcriptional regulator belonging to the zinc-cluster family. CGGNNNTANCGG was identified by PCR selection as the DNA sequence allowing its optimal binding. Nevertheless, this sequence is not a consensus sequence, the simultaneous presence of the two CGGs and the TA generally not being found in the known natural Cyp1p targets. In fact, our previous studies showed that the mechanism of Cyp1p DNA binding was target dependent. Data concerning the binding of Cyp1p to the UAS1-B/CYC1 are presented here. This target, containing the CGGGGTTTACGG sequence, was found to present the particular ability of stabilizing the binding of only one molecule of some monomeric Cyp1p fragments. This property was used to investigate the actual contribution of the TT and CGG sequences in the binding of Cyp1p. Our results indicate that each CGG belongs to a different half-site and, in contrast to a previous hypothesis, that the T nucleotide located four bases downstream from the left CGG is essential for the binding of one monomer to each half-site. The two half-sites of the UAS1-B/CYC1 thus overlap.

Carbon-Oxygen Lyases↗

PubMed1998

Sorting of D-lactate dehydrogenase to the inner membrane of mitochondria. Analysis of topogenic signal and energetic requirements.

D-Lactate dehydrogenase (D-LD) is located in the inner membrane of mitochondria. It spans the membrane once in an Nin-Cout orientation with the bulk of the protein residing as a folded domain in the intermembrane space. D-LD is synthesized as a precursor with an N-terminal cleavable presequence and is imported into the mitochondria in a Deltapsi-dependent, but mt-Hsp70-independent manner. Upon import in vitro D-LD folds in the intermembrane space to attain a conformation indistinguishable from endogenous D-LD. Sorting of D-LD to the inner membrane is directed by a composite topogenic signal consisting of the hydrophobic transmembrane segment and a cluster of charged amino acids C-terminal to it. We propose a model for the mode of operation of the sorting signal of D-LD. This model also accounts for the driving force of translocation across the outer membrane, in the apparent absence of mt-Hsp70-dependent assisted import and involves the folding of the D-LD in the intermembrane space.

Cell Membrane↗

PubMed1997

The ABC transporter Atm1p is required for mitochondrial iron homeostasis.

The function of the ABC transporter Atm1p located in the mitochondrial inner membrane is not yet known. To study its cellular role, we analyzed a mutant in which ATM1 was disrupted. Delta atm1 cells are deficient in the holoforms, but not the apoforms of heme-carrying proteins both within and outside mitochondria, yet both synthesis and transport of heme are functional. Delta atm1 cells are hypersensitive for growth in the presence of oxidative reagents, and they contain increased levels of the antioxidant glutathione, in particular of its oxidized form. Mitochondria deficient in Atm1p accumulate 30-fold higher levels of free iron as compared to wild-type organelles, i.e. three-fold more than mitochondria deficient in frataxin, the protein mutated in Friedreich's ataxia. The increased mitochondrial iron content may be causative of the oxidative damage of heme-containing proteins in delta atm1 cells. Our data assign an important function to Atm1p in mitochondrial iron homeostasis.

ATP-Binding Cassette Transporters↗

PubMed1997

The sorting route of cytochrome b2 branches from the general mitochondrial import pathway at the preprotein translocase of the inner membrane.

Cytochrome b2 is synthesized in the cytosol with a bipartite presequence. The first part of the presequence targets the protein to mitochondria and mediates translocation into the mitochondrial matrix compartment; the second part contains the sorting signal that is required for delivery of the protein to the intermembrane space. The localization of the structures that recognize the sorting signal is unclear. Here we show that upon import in vivo, the sorting signal of cytochrome b2 causes an early divergence from the general matrix import pathway and thereby prevents translocation of a folded C-terminal domain into mitochondria. By co-immunoprecipitations we find that translocation intermediates of cytochrome b2 are associated with Tim23, a component of the inner membrane protein import machinery. Cytochrome b2 constructs with an alteration in the sorting signal are mistargeted to the matrix of wild-type mitochondria. In mitochondria containing a mutant form of Tim23, however, the translocation of the altered sorting signal across the inner membrane is inhibited, and cytochrome b2 is correctly sorted to the intermembrane space. We suggest that the sorting signal of cytochrome b2 is recognized within the inner membrane in close vicinity to Tim23.

Biological Transport↗