Mitochondrial genes inPodospora anserina: Recombination and linkage

Summary A fifth cytoplasmic mutation (cap ^r 1) obtained inPodospora anserina is described. In addition to chloramphenicol resistance it confers a strong deficiency in cytochrome aa₃ and impairs the germination of ascospores. Genetic analysis shows: 1) strict maternal inheritance of (cap ^r 1) allele; 2) selection against the (cap ^r 1) allele as well in sexual crosses as during vegetative growth; 3) complete reversion of this selection by even low concentration of CAP. On the basis of their cytoplasmic inheritance and altered cytochrome spectra the five cytoplasmic mutations are assumed to be mitochondrial. Analysis of crosses between them allows to class them in 3 loci, 2 of which being closely linked.     

Polyprenyl quinones and α-tocopherol in Calendula officinalis

In various cellular subfractions of Calendula officinalis leaves a study was made of the distribution of polyprenyl quinones and α-tocopherol and the dynamics of their labelling with ¹⁴CO₂ and acetate-[1-¹⁴C] and incorporation of mevalonate-[2-¹⁴C] after 3 hr. It was confirmed that plastoquinone occurs only in the chloroplasts, ubiquinone only in the mitochondria and α-tocopherol in both these subfractions. Phylloquinone was found in the chloroplast and mitochondrial fractions as well as in the post-mitochondrial supernatant. Studies of the dynamics of radioactive precursor incorporation indicated that α-tocopherol is metabolized more rapidly than the polyprenyl quinones studied; the incorporation of mevalonate-[2-¹⁴C] suggests that the side chain of plastoquinone can be synthesized in the cytoplasm and transported to the chloroplasts.     

Calcium-Induced Alteration of Mitochondrial Morphology and Mitochondrial-Endoplasmic Reticulum Contacts in Rat Brown Adipocytes

Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca²⁺]_i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ^® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ^® drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca²⁺ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ^® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ^® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca²⁺ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ^® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes.Key words: Brown adipocyte, mitochondrial dynamics, calcium, endoplasmic reticulum     

Products of rat liver mitochondrial protein synthesis: electrophoretic analysis of the number and size of these proteins and their solubility in chloroform: methanol

Rat liver mitochondria were incubated in vitro with radioactive leucine, and submitochondrial particles prepared by several methods. Analysis of the labeled mitochondrial membrane fractions by sodium dodecylsulfate gel electrophoresis revealed three labeled bands of molecular weights corresponding to 40,000; 27,000; and 20,000 daltons. Electrophoresis for longer times at higher concentrations of acrylamide revealed eight labeled bands, ranging in molecular weights from 48,000 to 12,000.Mitochondria were incubated for 5 min with [³H]leucine followed by a chase of unlabeled leucine. Gel electrophoresis of the membranes obtained after labeling for 5 min indicated significant synthesis of polypeptides in the 40,000 M_r, range and very little labeling of low molecular-weight polypeptides. After addition of the chase, increased synthesis of the high molecular-weight polypeptides was observed; however, no significant increase or decrease of radioactivity in the bands of low molecular-weight was observed, suggesting that rat liver mitochondria have the ability to synthesize complete proteins in the M_r 27,000–40,000 range.Approximately 16% of the total leucine incorporated into protein by isolated rat liver mitochondria in vitro could be extracted by chloroform: methanol. Gel electrophoresis of the chloroform: methanol extract revealed several bands containing radioactivity with the majority of counts in a band of 40,000 molecular weight. Gel electrophoresis of the chloroform: methanol extract of lyophilized submitochondrial particles indicated label in two broad bands in the low molecular-weight region of 14,000-10,000 with insignificant counts in the higher molecular-weight regions of the gel.Yeast cells were pulse labeled in vivo with [³H]leucine in the presence of cycloheximide and the submitochondrial particles extracted with chloroform:methanol. The extract separated after gel electrophoresis into four labeled bands ranging in molecular weight from 52,000 to 10,000. Preincubation of the yeast cells with chloramphenicol prior to the pulse labeling caused a 6-fold stimulation of labeling into the band of lowest molecular weight of the chloroform: methanol extract. These results suggest that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins which are soluble in chloroform: methanol.     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

Temporary Sequestration of Potassium by Mitochondria in Astrocytes

Increases in extracellular potassium concentration ([K⁺]_o), which can occur during neuronal activity and under pathological conditions such as ischemia, lead to a variety of potentially detrimental effects on neuronal function. Although astrocytes are known to contribute to the clearance of excess K⁺_o, the mechanisms are not fully understood. We examined the potential role of mitochondria in sequestering K⁺ in astrocytes. Astrocytes were loaded with the fluorescent K⁺ indicator PBFI and release of K⁺ from mitochondria into the cytoplasm was examined after uncoupling the mitochondrial membrane potential with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Under the experimental conditions employed, transient applications of elevated [K⁺]_o led to increases in K⁺ within mitochondria, as assessed by increases in the magnitudes of cytoplasmic [K⁺] ([K⁺]_i) transients evoked by brief exposures to CCCP. When mitochondrial K⁺ sequestration was impaired by prolonged application of CCCP, there was a robust increase in [K⁺]_i upon exposure to elevated [K⁺]_o. Blockade of plasmalemmal K⁺ uptake routes by ouabain, Ba²⁺, or a mixture of voltage-activated K⁺ channel inhibitors reduced K⁺ uptake into mitochondria. Also, reductions in mitochondrial K⁺ uptake occurred in the presence of mito-K_ATP channel inhibitors. Rises in [K⁺]_i evoked by brief applications of CCCP following exposure to high [K⁺]_o were also reduced by gap junction blockers and in astrocytes isolated from connexin43-null mice, suggesting that connexins also play a role in K⁺ uptake into astrocyte mitochondria. We conclude that mitochondria play a key role in K⁺_o handling by astrocytes.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

Rabbit heart mitochondrial hexokinase: Solubilization and general properties

In rabbit heart, results show that two isoenzymes of hexokinase (HK) are present. The enzymatic activity associated with mitochondria consists of only one isoenzyme; according to its electrophoretic mobility and its apparent K_m for glucose (0.065 mm), it has been identified as type I isoenzyme. The bound HK I exhibits a lower apparent K_m for ATPMg than the solubilized enzyme, whereas the apparent K_m for glucose is the same for bound and solubilized HK. Detailed studies have been performed to investigate the interactions which take place between the enzyme and the mitochondrial membrane. Neutral salts efficiently solubilize the bound enzyme. Digitonin induces only a partial release of the enzyme bound to mitochondria; this result could be explained by the existence of contacts between the outer and the inner mitochondrial membranes [C. R. Hackenbrock (1968)Proc. Natl. Acad. Sci. USA61, 598–605]. Furthermore, low concentrations (0.1 mm) of glucose 6-phosphate (G6P) or ATP^4? specifically solubilize hexokinase. The solubilizing effect of G6P and ATP^4?, which are potent inhibitors of the enzyme, can be prevented by incubation of mitochondria with P_i or Mg²⁺. In addition, enzyme solubilization by G6P can be reversed by Mg²⁺ only when the proteolytic treatment of the heart homogenate is omitted during the course of the isolation of mitochondria. These results concerning the interaction of rabbit heart hexokinase with the outer mitochondrial membrane agree with the schematic model proposed by Wilson [(1982) Biophys. J.37, 18–19] for the brain enzyme. This model involves the existence of two kinds of interactions between HK and mitochondria; a very specific one with the hexokinase-binding protein of the outer mitochondrial membrane, which is suppressed by glucose 6-phosphate, and a less specific, cation-mediated one.     

[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]²⁺ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]²⁺ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]²⁺ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]²⁺ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]²⁺ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]²⁺ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.     

Endogenous Protein Phosphorylation in Rat Brain Mitochondria: Occurrence of a Novel ATP-Dependent Form of the Autophosphorylated Enzyme Succinyl-CoA Synthetase

Abstract: When rat brain mitochondria are incubated with [γ-³²P]ATP, there is a rapid (10 s) phosphorylation of proteins designated E, and F of M.W. 42,000 and 32,000, respectively. Although [γ-³²P]ATP was the preferred substrate for protein F, a small amount of labeling did occur with [γ-³²P]GTP. Phosphorylation of E₁ was absolutely ATP-dependent. On the other hand, a 32,000 M.W. protein from rat liver mitoplasts (mitochondria devoid of an outer membrane) was highly phosphorylated when [γ-³²P]GTP was used but not at all phosphorylated within short time periods with [γ-³²P]ATP. Both the ATP-labeled brain phosphoprotein F and GTP-labeled liver protein migrated to identical positions on high-resolution two-dimensional polyacrylamide gels, and both contained acid-labile phosphoryl groups. Furthermore, both phosphoproteins were identified as the autophosphorylated subunit of succinyl-CoA synthetase (SCS, EC 6.2.1.4) by using antibody directed against purified GTP-dependent porcine SCS. However, immunotitration experiments with anti-porcine SCS revealed that ATP- and GTP-labeled protein F in brain differed in their interactions with antibody, suggesting that in rat brain mitochondria two different forms of the enzyme exist that are immunologically distinct and differ in substrate specificity. When mitochondrial preparations enriched in particular brain cell or subcellular types were examined, an unequal distribution of E₁ and the two forms of protein F were observed. A brain subfraction containing neuronal cell body and glial mitochondria (CM) was found to contain E₁ and approximately equal amounts of the ATP- and GTP-dependent forms of protein F. Light synaptic mitochondria(SM₁) contained ATP-dependent protein F almost exclusively and were depleted in E₁. Dense synaptic mitochondria (SM₂) are rich in the ATP form of SCS but also contain low amounts of the GTP enzyme.     

Plant mitochondria use two pathways for the biogenesis of tRNAHis

All tRNA^His possess an essential extra G_–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G_–1 is added by a tRNA^His guanylyl transferase. In prokaryotes, G_–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G_–1 we find here that both maturation pathways can be used. Indeed, tRNA^His with or without a G_–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA^His precursors at both positions G₊₁ and G_–1. The G_–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA^His without G_–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA^His with a G_–1 are recovered. This shows that a previously unreported tRNA^His guanylyltransferase activity is present in plant mitochondria.     

Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

The transfer of Ca²⁺ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) can be excellently quantified with the ratiometric Ca²⁺ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ sensors, the cameleons, are efficiently used to specifically measure Ca²⁺ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca²⁺]_cyto and mitochondrial Ca²⁺ ([Ca²⁺]_mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca²⁺ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca²⁺]_cyto and [Ca²⁺]_mito within same individual cells. Our data indicate that depending on the kinetics of [Ca²⁺]_cyto rises there is a significant lag between onset of [Ca²⁺]_cyto and [Ca²⁺]_mito signals, pointing to a certain threshold of [Ca²⁺]_cyto necessary to activate mitochondrial Ca²⁺ uptake. The temporal correlation between [Ca²⁺]_mito and [Ca²⁺]_cyto as well as the efficiency of the transfer of Ca²⁺ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca²⁺ extrusion and a desensitization of mitochondrial Ca²⁺ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca²⁺ oscillations of pancreatic beta-cells in response to D-glucose.     

Mitochondrial S-adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans

S-adenosylmethionine (SAM), generated from methionine and ATP by S-adenosyl methionine synthetase (SAMS), is the universal methyl group donor required for numerous cellular methylation reactions. In Caenorhabditis elegans, silencing sams-1, the major isoform of SAMS, genetically or via dietary restriction induces a robust mitochondrial unfolded protein response (UPR^mt) and lifespan extension. In this study, we found that depleting SAMS-1 markedly decreases mitochondrial SAM levels. Moreover, RNAi knockdown of SLC-25A26, a carrier protein responsible for transporting SAM from the cytoplasm into the mitochondria, significantly lowers the mitochondrial SAM levels and activates UPR^mt, suggesting that the UPR^mt induced by sams-1 mutations might result from disrupted mitochondrial SAM homeostasis. Through a genetic screen, we then identified a putative mitochondrial tRNA methyltransferase TRMT-10C.2 as a major downstream effector of SAMS-1 to regulate UPR^mt and longevity. As disruption of mitochondrial tRNA methylation likely leads to impaired mitochondrial tRNA maturation and consequently reduced mitochondrial translation, our findings suggest that depleting mitochondrial SAM level might trigger UPR^mt via attenuating protein translation in the mitochondria. Together, this study has revealed a potential mechanism by which SAMS-1 regulates UPR^mt and longevity.     

Cross-compartment protection by SOD1

The absence of SOD1 in yeast has been found (M.A. Wallace et al., J. Biol. Chem. 279:32055–32062; 2004) to result in inactivation of Lys4p. This [4Fe-4S]-containing dehydratase is in the pathway of biosynthesis of lysine, hence the oxygen-dependent lysine auxotrophy seen in this case. O₂⁻ is known to oxidize and thus destabilize the [Fe–4S] clusters of dehydratases; hence, this would make perfect sense were it not for the fact that SOD1 localizes to the cytosol and the intermembrane space of mitochondria, whereas Lys4p localizes to the mitochondrial matrix. How could SOD1 in one compartment protect against O₂⁻ attack in a different compartment? We suggest that the relatively high levels of O₂⁻ in the cytosol and intermembrane space of the SOD1 mutant may react with endogenous NO, forming HOONO that can diffuse into the mitochondrial matrix and there inactivate Lys4p and other [4Fe–4S]-containing dehydratases.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

Coupling of the α1-adrenergic receptor to a guanine nucleotide-binding regulatory protein by a discrete domain distinct from its ligand recognition site

At rat hepatic membrane α₁-adrenergic receptors, the nonhydrolyzable GTP analogue p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding. This p[NH]ppG effect is consistent with the involvement of a guanine nucleotide-binding regulatory protein (G-protein) in α₁-adrenergic receptor signalling. Although readily apparent in membranes prepared to avoid retention of endogenous nucleotides and activation of Ca²⁺-sensitive proteinases (+pi), this p[NH]ppG effect is not observed in membranes prepared without proteinase inhibitors (−pi), or in −pi membranes treated with Ca²⁺ (−pi, +Ca²⁺). In these various membrane preparations, different M_r forms of the receptor are also identified by photoaffinity labelling with [¹²⁵I]CP65 526, an aryl azide analog of the α₁-selective antagonist, prazosin, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Whereas a predominant M_r = 80 000 subunit is identified in +pi membranes, in −pi membranes a proteolytic M_r = 59 000 fragment is also observed. In −pi, +Ca²⁺ membranes, only this latter peptide is detected. To evaluate the ability of each of these forms of the receptor to couple with a G-protein, the effect of p[NH]ppG on the agonist-inhibition of [¹²⁵I]CP65 526 labelling was determined by laser densitometry scanning and computer analysis. At the M_r = 80 000 subunit, p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding, even in −pi membranes. By contrast, agonist-binding at the M_r = 59 000 subunit is of low-affinity and was not affected by p[NH]ppG. These data indicate that the cleaved M_r = 59 000 fragment, while retaining hormone binding activity is unable to undergo G-protein coupling. Thus, the α₁-adrenergic receptor appears to contain a discrete domain necessary for G-protein coupling that is distinct from its ligand recognition site.     

Ultrastructure,composition of neutral lipids and their fatty acids of Candida tropicalis strain D-2 mutants resistant to the polyene antibiotic nystatin

This report deals with data on the cell ultrastructure of Candida tropicals strain D-2 mutants resistant to the polyene antibiotic, nystatin, and with an analysis of the fractional composition of neutral lipids and their fatty acids. The ultrastructural organization of the mutant cells is characterized by thickening of the cell wall and formation of invaginations into the cytoplasm, the appearance of new formations, large vacuoles, and reduction of the system of mitochondrial cristae. Lipids of nys^r mutants differ from those of the nys^s variant in having a decreased content of steroids and some fractions of neutral lipids. Certain nys^r mutants manifest difference in the relative amounts of saturated and unsaturated fatty acids (C_16:0, C_16:1, C_18:0, C_18:1).     

Identification of mitochondrial proteins on two-dimensional electrophoresis gels of extracts of adult Drosophila melanogaster

Several hundred proteins have been resolved on two-dimensional gels of extracts of [³⁵S]methionine-labeled adult Drosophila melanogaster. 27 of these polypeptides disappear from the gel pattern after feeding the K⁺ ionophore nonactin. These proteins have been identified as mitochondrial, since the two-dimensional gel pattern of extracts of isolated mitochondria correlates well with the pattern of the proteins missing from that of nonactin-treated flies. Nine new proteins also appear on the two-dimensional gels of the extracts from the nonactin-treated flies. Apparently, these nine proteins are precursors of the mature mitochondrial forms. These particular data support the concept that processing of many of the cytoplasmically synthesized mitochondrial proteins requires a specific membrane potential, and that some of these proteins are modified intramitochondrially. However, using [³⁵S]methionine incorporation techniques, not all labeled polypeptides disappear from mitochondria during such treatment. Feeding similarly radiolabeled flies with chloramphenicol, an inhibitor of mitochondrial protein synthesis, results in the disappearance of only one protein from the gel pattern with the concurrent appearance of a ‘new’ high-molecular-weight polypeptide. Collectively, these data show that a specific group of [³⁵S]methionine-labeled mitochondrial proteins can be identified by selective inhibition of mitochondrial function in whole cell protein maps of adult D. melanogaster.