The S. Cerevisiae HAP complex, a key regulator of mitochondrial function, coordinates nuclear and mitochondrial gene expression.

We have compared Saccharomyces cerevisiae global gene expression in wild-type and mutants (Deltahap2 and Deltahap4) of the HAP transcriptional complex, which has been shown to be necessary for growth on respiratory substrates. Several hundred ORFs are under positive or negative control of this complex and we analyse here in detail the effect of HAP on mitochondria. We found that most of the genes upregulated in the wild-type strain were involved in organelle functions, but practically none of the downregulated ones. Nuclear genes encoding the different subunits of the respiratory chain complexes figure in the genes more expressed in the wild-type than in the mutants, as expected, but in this group we also found key components of the mitochondrial translation apparatus. This control of mitochondrial translation may be one of the means of coordinating mitochondrial and nuclear gene expression in elaborating the respiratory chain. In addition, HAP controls the nuclear genes involved in several other mitochondrial processes (import, mitochondrial division) that define the metabolic state of the cell, but not mitochondrial DNA replication and transcription. In most cases, a putative CCAAT-binding site is present upstream of the ORF, while in others no such sites are present, suggesting the control to be indirect. The large number of genes regulated by the HAP complex, as well as the fact that HAP also regulates some putative transcriptional activators of unknown function, place this complex at a hierarchically high position in the global transcriptional regulation of the cell.

Expression and localization of hepatocyte domain-specific plasma membrane proteins in hepatoma x fibroblast hybrids and in hepatoma dedifferentiated variants.

We have studied two aspects of the plasma membrane of hepatocytes, highly differentiated epithelial cells that exhibit a particular and complex polarity. Using a genetic approach, we have distinguished between the expression/regulation of proteins specific for all three hepatocyte membrane domains and their organization into discrete domains. For this analysis we used a panel of previously isolated cell clones, derived from the differentiated rat hepatoma line H4IIEC3, and that present different expression patterns for liver-specific genes. This panel was composed of (1) differentiated clones, (2) chromosomally reduced hepatoma-fibroblast hybrids characterized by a pleiotropic extinction/reexpression of liver-specific genes and (3) dedifferentiated variant and revertant clones. The expression of 16 hepatocyte membrane polarity markers was studied by western blotting and immunolocalization. Even though cells of differentiated clones express all of these polarity markers, they are not polarized, and are therefore suitable for studying the regulation of plasma membrane protein expression, and for identifying gene products implicated in the establishment of membrane polarity. In hepatoma-fibroblast hybrids the expression of four markers, three apical (dipeptidylpeptidase IV, alkaline phosphodiesterase B10 and polymeric IgA receptor) and one lateral (E-cadherin), is down-regulated in extinguished clones and restored in reexpressing subclones, as previously reported for liver-specific functions. The dipeptidylpeptidase IV mRNA was undetectable or strongly reduced in extinguished hybrids, but expressed at a robust level in some of the reexpressing clones. Concerning the dedifferentiated variants, each has its own pattern of membrane marker expression (loss of expression of three to six markers), that differs from that of extinguished hybrids. Revertant cells express all of the membrane markers examined. Among all of these hepatoma derivatives, only cells of reexpressing hybrids are polarized, and form bile canaliculi-like structures, with spherical and even, for one clone, long tubular and branched forms. All apical markers examined are confined in these canalicular structures, whereas the other markers are excluded from them, and present on the rest of the membrane (basolateral markers) or at the cell-cell contacts (lateral markers). Cells of reexpressing hybrids also express simple epithelial polarity. Thus the expression of only a few hepatocyte-domain-specific plasma membrane proteins is subject to down-regulation, as is the case for liver-specific genes so far studied, and the expression of polarity markers and the formation of poles are dissociable events.

Function-directed mutagenesis of the cytochrome b6f complex in Chlamydomonas reinhardtii: involvement of the cd loop of cytochrome b6 in quinol binding to the Q(o) site.

The FUD2 mutant from the green alga Chlamydomonas reinhardtii expresses a cytochrome b6 variant of higher apparent molecular mass [Lemaire et al. (1986) Biochim. Biophys. Acta 851, 239-248]. Here, we show that the mutation corresponds to a 36 base pair duplication in the chloroplast petB gene, which corresponds to a 12 amino acid duplication in the cd loop of cytochrome b6. The resulting protein still binds its heme cofactors and assembles into cytochrome b6f complexes, which accumulate in wild type amounts in exponentially growing cells of FUD2. However, these cytochrome b6f complexes show loosened binding of the Rieske protein and are more prone to degradation in aging cells. Electron transfer through the cytochrome b6f complexes is about 8 times slower in FUD2 than in wild type cells. This is due to a slower oxidation of plastoquinol at the Q(o) site, the folding of which is most likely altered by the duplication. By varying the redox state of the plastoquinone pool in vivo, we show that there is a dramatic decrease in the affinity of the Q(o) site for plastoquinols, which is about 100 times lower in FUD2 than in wild type cells. Our results show that the value of the binding constant of plastoquinol to the Q(o) site (2 x 10(4) M(-1)) derived in [Kramer et al. (1994) Biochim. Biophys. Acta 1184, 251-262] may be extrapolated to in vivo conditions.

Molecular genetic identification of a pathway for heme binding to cytochrome b6.

Heme binding to cytochrome b6 is resistant, in part, to denaturing conditions that typically destroy the noncovalent interactions between the b hemes and their apoproteins, suggesting that one of two b hemes of holocytochrome b6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b6, and to identify mutants that are blocked at one step of this pathway. Chlamydomonas reinhardtii strains carrying substitutions in either one of the four histidines that coordinate the bh or bl hemes to the apoprotein were created. These mutations resulted in the appearance of distinct immunoreactive species of cytochrome b6, which allowed us to specifically identify cytochrome b6 with altered bh or bl ligation. In gabaculine-treated (i.e. heme-depleted) wild type and site-directed mutant strains, we established that (i) the single immunoreactive band, observed in strains carrying the bl site-directed mutations, corresponds to apocytochrome b6 and (ii) the additional band present in strains carrying bh site-directed mutations corresponds to a bl-heme-dependent intermediate in the formation of holocytochrome b6. Five nuclear mutants (ccb strains) that are defective in holocytochrome b6 formation display a phenotype that is indistinguishable from that of strains carrying site-directed bh ligand mutants. The defect is specific for cytochrome b6 assembly, because the ccb strains can synthesize other b cytochromes and all c-type cytochromes. The ccb strains, which define four nuclear loci (CCB1, CCB2, CCB3, and CCB4), provide the first evidence that a b-type cytochrome requires trans-acting factors for its heme association.

Maturation of pre-apocytochrome f in vivo. A site-directed mutagenesis study in Chlamydomonas reinhardtii.

The biosynthesis of cytochrome f is a multistep process which requires processing of the precursor protein and covalent ligation of a c-heme upon membrane insertion of the protein. The crystal structure of a soluble form of cytochrome f has revealed that one axial ligand of the c-heme is provided by the alpha-amino group of Tyr1 generated upon cleavage of the signal sequence from the precursor protein (Martinez S. E., Huang D., Szczepaniak A., Cramer W.A., and Smith J. L. (1994) Structure 2, 95-105). We therefore investigated, by site-directed mutagenesis, the possible interplay between protein processing and heme attachment to cytochrome f in Chlamydomonas reinhardtii. These modifications were performed by chloroplast transformation using a petA gene encoding the full-length precursor protein and also a truncated version lacking the C-terminal membrane anchor. We first substituted the two cysteinyl residues responsible for covalent ligation of the c-heme, by a valine and a leucine, and showed that heme binding is not a prerequisite for cytochrome f processing. In another series of experiments, we replaced the consensus cleavage site for the thylakoid processing peptidase, AQA, by an LQL sequence. The resulting transformants were nonphototrophic and displayed delayed processing of the precursor form of cytochrome f, but nonetheless both the precursor and processed forms showed heme binding and assembled in cytochrome b6f complexes. Thus, pre-apocytochrome f adopts a suitable conformation for the cysteinyl residues to be substrates of the heme lyase and pre-holocytochrome f folds in an assembly-competent conformation. In the last series of experiments, we compared the rates of synthesis and degradation of the various forms of cytochrome f in the four types of transformants under study: (i) the C terminus membrane anchor apparently down-regulates the rate of synthesis of cytochrome f and (ii) degradation of misfolded forms of cytochrome f occurs by a proteolytic system intimately associated with the thylakoid membranes.

The petD gene is transcribed by functionally redundant promoters in Chlamydomonas reinhardtii chloroplasts.

FUD6, a nonphotosynthetic mutant of Chlamydomonas reinhardtii, was previously found to be deficient in the synthesis of subunit IV of the cytochrome b6/f complex, the chloroplast petD gene product (C. Lemaire, J. Girard-Bascou, F.-A. Wollman, and P. Bennoun, Biochim. Biophys. Acta 851:229-238, 1986). The lesion in FUD6 is a 236-bp deletion between two 11-bp direct repeats in the chloroplast genome. It extends from 82 to 72 bp upstream of the 5' end of wild-type petD mRNA to 156 to 166 bp downstream of the 5' end. Thus, the deletion extends into the putative promoter and 5' untranslated region of petD. No petD mRNA of the normal size can be detected in FUD6 cells, but a low level of a dicistronic message accumulates, which contains the coding regions for subunit IV and cytochrome f, the product of the upstream petA gene. petD transcriptional activity in FUD6 is not significantly altered from the wild-type level. This transcriptional activity was eliminated by petA promoter disruptions, suggesting that it originates at the petA promoter. We conclude that the petD-coding portion of most cotranscripts is rapidly degraded in FUD6, possibly following processing events that generate the 3' end of petA mRNA. A chloroplast transformant was constructed in which only the sequence from -81 to -2 relative to the major 5' end of the petD transcript was deleted. Although this deletion eliminates all detectable petD promoter activity, the transformant grows phototrophically and accumulates high levels of monocistronic petD mRNA. We conclude that the petD gene can be transcribed by functionally redundant promoters. In the absence of a functional petD promoter, a lack of transcription termination allows the downstream petD gene to be cotranscribed with the petA coding region and thereby expressed efficiently.

Nucleotide sequences of the continuous and separated petA, petB and petD chloroplast genes in Chlamydomonas reinhardtii.

We have mapped and sequenced the petA (cytf), petB (cytb6) and petD (subunit IV) genes on the chloroplast genome of Chlamydomonas reinhardtii. At variance with the pet genes in higher plant chloroplasts, the petB and petD genes are continuous, not adjacent and not located next to the psbB gene. The corresponding polypeptide sequences are highly conserved when compared with their counterparts from other sources but have a few features specific of algal cytb6/f complexes. In particular the transit sequence of cytf displays unique characteristics when compared with those previously described for cytf in higher plants.

Incorporation of bacteriorhodopsin into large unilamellar liposomes by reverse phase evaporation.

The reverse phase evaporation procedure was used to prepare large unilamellar liposomes containing bacteriorhodopsin. Electron microscopy showed that proteoliposomes were unilamellar and fairly uniform in size provided the preparation was extruded through calibrated nucleopore membranes : the vesicles have diameters around 200 nm. The spectral properties of the bacteriorhodopsin in the large liposomes resembled those of bacteriorhodopsin in purple membrane. Furthermore, the chromoprotein in the reconstituted vesicles had an inside-out orientation and on illumination, translocated protons efficiently from the external medium into the vesicles in the presence of the ionophore valinomycin. In the absence of the latter, a light-independent transmembrane potential of about 60 mV was measured from thiocyanate distribution. In the presence of valinomycin, this transmembrane electrical potential was abolished and then a light-dependent transmembrane pH gradient of about 2 pH units could be generated.

Pressure-induced inactivation of sarcoplasmic reticulum adenosine triphosphatase during high-speed centrifugation.

Sarcoplasmic reticulum vesicles were found to be highly sensitive to high-speed centrifugation in metal-deprived mediums at low temperature (4 degrees C). The irreversible modifications induced were easily detected from observation of the environment-sensitive spectrum of an iodoacetamide spin-label bound to the ATPase. Centrifugation also resulted in vesicle aggregation and inhibition of calcium transport, ATPase activity, and phosphoenzyme formation. These denaturation-like phenomena were prevented in the presence of sucrose, or by nucleotide binding, or, again, by cation binding to the ATPase high-affinity calcium binding sites and were only present when centrifugation was performed at low temperature. The crucial parameter during this process was found to be the hydrostatic pressure which developed in the centrifuge tube. SR vesicles exposed to 800 bars in a pressure bomb displayed the same features. It is suggested that irreversible denaturation takes place after one or both of the two following well-documented effects of pressure: a rise in the lipid order/disorder transition temperature or dissociation of the oligomeric structure of the calcium pump.

A direct fluorescence study of the transient steps induced by calcium binding to sarcoplasmic reticulum ATPase.

The sarcoplasmic reticulum intrinsic fluorescence level was closely correlated with the ATPase functional state, from pH 5.5 to 8.5. The fluorescence signal was used in stopped flow measurements for direct study of transient pump kinetics after calcium binding or removal. The signal change time course, which depends solely on the free calcium concentration in the observation chamber, was analyzed as a single exponential. Rate constants (kobs) were relatively slow (5 to 20 s-1), indicating multistep interaction between calcium and the transport protein. At pH 7 and 20 degrees C, and in the presence of 100 mM potassium and 1 to 20 mM MgCl2, kobs first decreased, and then increased as the calcium concentration rose. Similar experiments were performed at pH 6. Data were analyzed according to a scheme in which sarcoplasmic reticulum . calcium complex formation is controlled by a slow isomerization step occurring either before or after the rapid calcium binding to the high affinity site. The results are discussed with reference to published rapid quenching experiments. Under our conditions, i.e. in the absence of a calcium gradient across the membrane, the calcium pump cycle step in which reorientation of the calcium binding sites occurs cannot be identified with the isomerization step mentioned above.