The interaction of quinone analogues with wild-type and ubiquinone-deficient yeast mitochondria.

The interaction of the exogenous quinones, duroquinone (DQ) and the decyl analogue of ubiquinone (DB) with the mitochondrial respiratory chain was studied in both wild-type and a ubiquinone-deficient mutant of yeast. DQ can be reduced directly by NADH dehydrogenase, but cannot be reduced by succinate dehydrogenase in the absence of endogenous ubiquinone. The succinate-driven reduction of DQ can be stimulated by DB in a reaction inhibited 50% by antimycin and 70-80% by the combined use of antimycin and myxothiazol, suggesting that electron transfer occurs via the cytochrome b-c1 complex. Both DQ and DB can effectively mediate the reduction of cytochrome b by the primary dehydrogenases through center o, but their ability to mediate the reduction of cytochrome b through center i is negligible. Two reaction sites for ubiquinol seem to be present at center o: one is independent of endogenous Q6 with a high reaction rate and a high Km; the other is affected by endogenous Q6 and has a low reaction rate and a low Km. By contrast, only one ubiquinol reaction site was observed at center i, where DB appears to compete with endogenous Q6. DB can oxidize most of the pre-reduced cytochrome b, while DQ can oxidize only 50%. On the basis of these data, the possible binding patterns of DB on different Q-reaction sites and the requirement for ubiquinone in the continuous oxidation of DQH are discussed.

The role of the membrane-spanning and extra-membranous regions of the iron-sulfur protein in its assembly into the cytochrome bc1 complex of yeast mitochondria.

The assembly of six deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex was investigated by immunoprecipitation from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the intact cytochrome bc1 complex. After import, the mutant proteins lacking residues 41-55 or 66-78, located at the membrane-spanning region of the protein, and residues 182-196 located at the C-terminus of the protein, were assembled in vitro into the bc1 complex approximately 50% as effectively as the wild type iron-sulfur protein suggesting that these regions of the iron-sulfur protein may not be critical for the assembly. By contrast, only trace amounts of the mutant proteins lacking residues 80-95, 122-135, 138-153 located in the extra-membranous region of the iron-sulfur protein were assembled into the bc1 complex. After import in vitro into mitochondria isolated from a cytochrome b-deficient yeast strain, the mutants lacking residues 41-55 and 182-196 were assembled as efficiently as the wild type; however, the mutants lacking residues 55-66 and 66-78 were assembled less efficiently in the absence of cytochrome b suggesting that the hydrophobic membrane-spanning region, residues 55-78, of the iron-sulfur protein, may interact with cytochrome b during the assembly of the bc1 complex.

Mutations in the tether region of the iron-sulfur protein affect the activity and assembly of the cytochrome bc(1) complex of yeast mitochondria.

Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein. EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.

Oxidation of NADH by a rotenone and antimycin-sensitive pathway in the mitochondrion of procyclic Trypanosoma brucei brucei.

The pathway of NADH oxidation in the procyclic Trypanosoma brucei brucei was investigated in a crude mitochondrial membrane fraction and in whole cells permeabilized with digitonin. NADH:cytochrome c reductase activity was 75% inhibited by concentrations of antimycin that inhibited 95% succinate:cytochrome c reductase activity suggesting that the major pathway for NADH oxidation in the mitochondria involved the cytochrome bc1 complex of the electron transfer chain. Both NADH:cytochrome c and NADH:ubiquinone reductase activities were inhibited 80-90% by rotenone indicating the presence of a complex I-like NADH dehydrogenase in the mitochondrion of trypanosomes. In whole cells permeabilized with low concentrations of digitonin, the oxidation of malate, proline and glucose (in the presence of salicylhydroxamic acid, the inhibitor of the alternate oxidase) was inhibited 30-50% by rotenone. The presence of an alternative pathway for NADH oxidation involving fumarate reductase was indicated by the observation that malonate, the specific inhibitor of succinate dehydrogenase, inhibited 30-35% the rate of oxygen uptake with malate and glucose as substrates in the digitonin-permeabilized cells. We conclude that in the mitochondrion of the procyclic form of T. brucei, NADH is preferentially oxidized by a rotenone-sensitive NADH:ubiquinone oxidoreductase; however, NADH can also be oxidized to some extent by the enzyme fumarate reductase present in the mitochondrion of T. brucei.

Expanding the view of scholarship: introduction.

The Association of American Medical Colleges' Council of Academic Societies (CAS) has a long-standing interest in scholarship as it relates to research, education, and service, the traditional definition of the activities of medical school. The work of Ernest Boyer and Charles Glassick is highly respected for redefining scholarship and conceiving how scholarship as thus defined can be assessed. Because their ideas have been applied in other areas of the academy but not widely in medical faculties, the CAS Task Force on Scholarship collected a special set of papers on Boyer's four areas of scholarship as applied to medical school, including case studies and the perspective from the university. The four areas of scholarship defined by Boyer and Glassick are the scholarship of discovery, the scholarship of integration, the scholarship of application, and the scholarship of teaching. The scholarship of discovery-research-has for decades been the primary focus for promotion and tenure for medical school faculty, even though the faculty also had major and critical activities in the other areas of scholarship. The CAS hopes that the ideas put forth in this special theme issue will produce a continuing dialogue as faculty and administrators at medical schools reflect on the value of these different forms of scholarship, their application by medical school faculty, and their contributions to the individual missions of each medical school and teaching hospital. In addition, these articles will stimulate continuing discussions that will definite equitable methods for the continued assessment of the scholarly accomplishments of medical school faculty.

Capturing the promise of science in medical schools.

To gain a better understanding of the effects on medical schools of transformations in medical practice, science, and public expectations, the Association of American Medical Colleges (AAMC) constituted the Advisory Panel on the Mission and Organization of Medical Schools (APMOMS) in 1994. APMOMS created six working groups to address the issues deemed by panel members to be of highest priority. This article is a report of the findings of the Working Group on Capturing the Promise of Medical Research, which addressed questions concerning the direction of research and the integration of scientific developments in medical education and practice. The working group explored a broad panorama of issues, including those related to sustaining the accomplishments, momentum, and progress of medical research. A dominant theme emerged: the central importance of an environment of discovery to the core missions of medical schools. The present article consists of the group's comments and recommendations on the main topic-the promise of biomedical research in relation to medical education-and their comments and recommendations on five other topics that have important relationships to the main topic and to the group's central charge. These are ethics; academia-industry relations; the administrative structure of medical schools; university-medical school relations; and research funding.

A proposed pathway of proton translocation through the bc complexes of mitochondria and chloroplasts.

The cytochrome bc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP. Proton translocation studies with radiolabeled N,N'-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50-70% with minimal effect on electron transfer in the cytochrome bc1 and cytochrome bf complexes reconstituted into liposomes. Subsequent binding studies with cytochrome bc1 and cytochrome bf complexes indicate that DCCD specifically binds to the subunit b and subunit b6, respectively, in a time and concentration dependent manner. Further analyses of the results with cyanogen bromide and protease digestion suggest that the probable site of DCCD binding is aspartate 160 of yeast cytochrome b and aspartate 155 or glutamate 166 of spinach cytochrome b6. Moreover, similar inhibition of proton translocating activity and binding to cytochrome b and cytochrome b6 were noticed with N-cyclo-N-(4-dimethylamino-napthyl)carbodiimide (NCD-4), a fluorescent analogue of DCCD. The spin-label quenching experiments provide further evidence that the binding site for NCD-4 on helix cd of both cytochrome b and cytochrome b6 is localized near the surface of the membrane but shielded from the external medium.

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes.

Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of(14)C-leucine or(14)C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of(3)H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of(14)C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to(14)C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to(14)C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.

Coenzyme Q analogues reconstitute electron transport and proton ejection but not the antimycin-induced "red shift" in mitochondria from coenzyme Q deficient mutants of the yeast Saccharomyces cerevisiae.

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase activity but contained normal amounts of cytochromes b and c1 by spectral analysis. Addition of the exogenous coenzyme Q derivatives including Q2, Q6, and the decyl analogue (DB) restored the rate of antimycin- and myxothiazole-sensitive cytochrome c reductase with both substrates to that observed with reduced DBH2. Similarly, addition of these coenzyme Q analogues increased 2-3-fold the rate of cytochrome c reduction in mitochondria from wild-type cells, suggesting that the pool of coenzyme Q in the membrane is limiting for electron transport in the respiratory chain. Preincubation of mitochondria from the Q-deficient yeast cells with DBH2 at 25 degrees C restored electrogenic proton ejection, resulting in a H+/2e- ratio of 3.35 as compared to a ratio of 3.22 observed in mitochondria from the wild-type cell. Addition of succinate and either coenzyme Q6 or DB to mitochondria from the Q-deficient yeast cells resulted in the initial reduction of cytochrome b followed by a slow reduction of cytochrome c1 with a reoxidation of cytochrome b. The subsequent addition of antimycin resulted in the oxidant-induced extrareduction of cytochrome b and concomitant oxidation of cytochrome c1 without the "red" shift observed in the wild-type mitochondria. Similarly, addition of antimycin to dithionite-reduced mitochondria from the mutant cells did not result in a red shift in the absorption maximum of cytochrome b as was observed in the wild-type mitochondria in the presence or absence of exogenous coenzyme Q analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome b is necessary for the effective processing of core protein I and the iron-sulfur protein of complex III in the mitochondria.

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in a mutant of yeast (W-267, Box 6-2) that lacks a spectrally detectable cytochrome b and synthesizes a shortened form of apocytochrome b. We recently reported that several cytochrome b-deficient mutants contained significantly diminished amounts of core proteins I and II as well as the iron-sulfur protein, but contained equal amounts of cytochrome c1 compared to the wild type (K. Sen and D. S. Beattie, Arch. Biochem. Biophys. 242, 393-401, 1985). In the present study, the time course of processing of precursors of both core protein I and the iron-sulfur protein which had accumulated in cells treated with the uncoupler carbonyl m-chlorophenyl hydrazone (CCCP) was noted to be significantly lower in the mutant compared to the wild type. The amounts of the mature forms of these proteins in mitochondria pulse labeled under different conditions was also considerably decreased at all times studied. The synthesis of both proteins appeared to be unaffected in the mutant, as the precursor forms of both proteins accumulated to the same extent when processing in vivo was blocked by CCCP. Furthermore, translation of RNA in a reticulocyte lysate in vitro indicated that the messenger RNAs for both proteins were present in the mutant and translated with equal efficiency. The import into isolated mitochondria of the precursor forms of the iron-sulfur protein synthesized in the cell-free system was also decreased in the mutant mitochondria. In addition, the precursor form was bound to the exterior of the mitochondrial membrane where it was sensitive to digestion with proteases. By contrast, the synthesis and processing of cytochrome c1 appeared to be unaffected in these mutants. These results suggest that cytochrome b is necessary for the proper processing and assembly of both core protein I and the iron-sulfur protein, but not for cytochrome c1, into complex III of the inner mitochondrial membrane.