Role of Heme in Synthesis and Membrane Binding of Succinic

Transcription

1 JOURNAL OF BACTERIOLOGY, May 1979, p Vol. 138, No /79/5-377/6$2./ Role of Heme in Synthesis and Membrane Binding of Succinic Dehydrogenase in Bacillus subtilis ERIK HOLMGREN, LARS HEDERSTEDT, AND LARS RUTBERG* Departnent of Bacteriology, Karolinska Institutet, S-14 1 Stockholn, Sweden Received for publication 13 November 1978 A 5-aminolevulinic acid-requiring mutant of Bacillus subtilis was isolated. When the mutant is shifted from medium containing 5-aminolevulinic acid to medium lacking this growth factor, the bacteria continued to grow at undiminished rate for about three generations. The membranes from these bacteria contained severely reduced amounts of cytochrome. The mutant was used to study the role of heme synthesis on synthesis and membrane binding of succinic dehydrogenase (SDH). The amount of SDH in whole-cell lysates in the soluble cytoplasmic fraction and in membranes was determined by one-dimensional (rocket) immunoelectrophoresis with an SDH-specific antiserum. After heme synthesis was blocked, the relative amount of SDH in the membrane decreased, whereas increasing amounts of SDH antigen were found in the cytoplasm. When heme synthesis was resumed on readdition of 5-aminolevulinic acid, the amount of membrane-bound SDH antigen increased at a much faster rate than net synthesis. During a 3-h growth period without 5-aminolevulinic acid, there was little change in the pattern of membrane proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled membranes, as compared to membranes from control cultures. However, both the 65,-dalton and the 28,-dalton polypeptides of the SDH complex (L. Hederstedt, E. Holmgren, and L. Rutberg, J. Bacteriol. 138:37-376, 1979) were present in decreasing amounts in membranes from 5-aminolevulinic acid-starved bacteria. From these results we suggest that SDH in B. subtilis is synthesized as a soluble protein and becomes membrane bound only when it attaches to a site in the membrane, (part of) which is a cytochrome of b type. Succinic dehydrogenase [SDH; EC , succinate:(acceptor) oxidoreductase] can be solubilized from the Bacillus subtilis cytoplasmic membrane with nonionic detergent, e.g., Triton X-1. In a previous paper we have shown that an enzymatically active, solubilized, SDH complex contains equimolar amounts of three polypeptides of approximate molecular weights (65,, 28,, and 19, (9). The two larger polypeptides are thought to constitute the enzyme proper, whereas the 19,-dalton polypeptide has been tentatively identified as cytochrome b. This leads to the suggestion that there is a structural relation in the membrane between SDH and cytochrome, e.g., cytochrome may be essential for binding of the enzyme to the cytoplasmic membrane. In the present work we have used a mutant auxotrophic for 5-aminolevulinic acid (1) to examine the possible relation between cytochrome and SDH synthesis. In the absence of 5-aminolevulinic acid, this mutant cannot make cytochrome. For similar mutants in Escherichia coli 377 (7) it has been suggested that the apoprotein is still synthesized and incorporated into the membrane when heme synthesis is blocked, whereas this does not seem to be the case for cytochrome cl in bakers' yeast (14). Which situation prevails in heme-deficient B. subtilis is not known. The results of the experiments to be presented indicate that there is close coupling between cytochrome synthesis and membrane binding of SDH. MATERIALS AND METHODS Bacteria. The B. subtilis strains employed are listed in Table 1. The 5-aminolevulinic acid-requiring mutant was isolated essentially as described by Anderson and Ivanovics (1). About 18 spores of BrlO2 were spread on TBAB plates containing 2,ug of streptomycin per ml. The plates were incubated at 37 C for 3 to 4 days, and the resulting colonies were tested for 5-aminolevulinic acid auxotrophy by streaking them on minimal casein hydrolysate plates with and without 5-aminolevulinic acid. Out of 25 colonies tested, 4 required 5-aminolevulinic acid for growth. DNA was extracted from one of these, designated

2 378 HOLMGREN, HEDERSTEDT, AND RUTBERG TABLE 1. Bacterial strains Strain Relevant properties Origin or derivation BrlO2 trpc2 hisb J. Spizizen 3G18 trpc2 met ade G. Venema Br95 trpc2pheai ilvcl J. Spizizen KA1 trpc2 hisb str 5-Ala BrlO2 KAll trpc2 met 5-Ala 3G18 (DNA from KA 1) KA1, and used to transform 3G18 to adenine prototrophy at saturating amounts of DNA. The Ade+ transformants were restreaked on the same medium and tested for 5-aminolevulinic acid requirement. One such recombinant was kept and designated KAll. KAll is not streptomycin resistant (1). The amount of 5-aminolevulinic acid added to different media was 2 ug/inl. Growth of bacteria. The general methods for growing the bacteria and for preparation of membranes and soluble fractions were as recently described (9, 15). The details of the experiments are given in the figure legends. For labeling of proteins, 1,uCi of a 1Clabeled L-amino acid mixture (New England Nuclear Corp.) was added per ml of medium. Other techniques. The methods used for antiserum preparations, SDH enzyme activity determination, immunoelectrophoresis, sodium dodecyl sulfatepolyacrylamide gel electrophoresis (slab gels), and autoradiography were as recently described (9, 15). RESULTS Properties of the 5-aminolevulinic acidrequiring mutant KAll. The mutant KAll was isolated by transforming strain 3G18 with saturating amounts of KA1 DNA with selection for adenine prototrophs on plates containing 5- aminolevulinic acid (congression). To get some information regarding the position of the 5-Ala mutation in KAll, limiting amounts of KAll DNA was used to transform Br95 and Br12, respectively. Amino acid prototrophs were selected on plates containing 5-aminolevulinic acid, and these transformants were then tested for the 5-aminolevulinic acid growth requirement. No linkage was found between 5-Ala and hisb (/64 transformants tested), whereas 4/ 161 Phe+ transformants and 26/134 Ilv+ transformants, respectively, required 5-aminolevulinic acid for growth. This result suggests that the 5-Ala mutation is in the hema locus (1). Optimnal growth of KAll in minimal casein hydrolysate medium was obtained at 2 ug or more of 5-aminolevulinic acid per ml. When KAll was shifted from a medium containing this growth factor to a medium lacking it, growth continued at undiminished rate for about three generations, with concomitant increase in the number of viable cells. Membrane preparations from KAll grown without 5-aminolevulinic acid are white compared to the reddish color of membranes from bacteria grown in supplemented medium, reflecting their low cytochrome content (Fig. 1). Effect ofblocking heme synthesis on SDH synthesis. The purpose of the following experiments was to study the role of heme synthesis on the synthesis and cellular location of SDH. The basic design of the experiments is similar. The 5-aminolevulinic acid-requiring mutant KAll is grown in minimal casein hydrolysate medium with and without this growth factor. Samples are then taken at intervals, and SDH is determined by measuring in vitro enzymatic activity and/or the amount of SDH antigen in rocket immunoelectrophoresis with SDH-specific antiserum. In the first experiments KA11 was grown with 5-aminolevulinic acid and then shifted to medium with or without the growth factor. Samples were taken every 3 min during a 3-h period, and membranes were prepared. The growth rate ofthe two cultures was identical during the first 12 min of the experiment, but after this time the growth rate declined more rapidly in the culture grown without 5-aminolevulinic acid (Fig. 2). The relative amount of SDH enzyme activity in the membrane, calculated per milliliter of sample, did not increase more than about 5% in the culture grown without 5-aminolevulinic acid, whereas the activity in the control culture increased about 1-fold (data not shown). It is known that the relative ~*X I D4 ' J. BACTERIOL n Wavelength FIG. 1. Reduced-minus-oxidized absorption spectra of membranes at room temperature. KAII was grown in medium containing 5-aminolevulinic acid, and then shifted to medium with and without the growth factor. Three generations after shift, membranes were prepared via protoplasts as described (15). Difference in absorption spectra was recorded as described (9) on an Aminco-Chance double-beam spectrophotometer. Membranes were from bacteria grown in the presence of 5-aminolevulinic acid (, 5. mg ofprotein per ml) and in the absence of the growth factor (- - -, 5.9 mg ofprotein per ml).

3 VOL. 138, ov - I MINUTES FIG. 2. Strain KAI1 was grown in minimal casein hydrolysate medium with 2 pg of tryptophan, 2 pg of methionine, and 2 pg of 5-aminolevulinic acid per ml, respectively, in 1,6-ml Fernbach flasks at 37 C with shaking to an absorbance at 6 nm (A66) of.4. The cells were centrifuged, and the pellets were suspended in 25 ml of medium without 5-aminolevulinic acid. To 3 ml of medium with and without 5-aminolevulinic acid was added 1 ml bacteria, the cultures were incubated at 37 C with shaking, and 5- ml samples were collected at intervals. The samples were centrifuged, and the bacteria were stored at -2 C overnight. The next day the pellets were thawed and washed twice with.1 Mpotassium phosphate buffer (ph 8.) and finally suspended in 1 ml of buffer with 1 mm phenylmethylsulfonylfluoride. To each sample was added.1 ml of lysozyme (3 mg/ml),.1 ml ofdnase and RNase (1X)g/ml) and.1 ml of 1 mm MgSO4. The samples were incubated at 37 C for 1 h. Membranes were then prepared and run in rocket immunoelectrophoresis. Protein was determined by a modification of the method of Lowry et al. (9). Symbols: *, *, with 5-aminolevulinic acid; E,, without 5-aminolevulinic acid; *, O, growth of bacteria; *,, membrane protein. amount of SDH increases as B. subtilis approaches stationary phase (13). The amount of SDH antigen measured in rocket immunoelectrophoresis is reduced in membranes from bacteria grown without 5-aminolevulinic acid (Fig. 2). The results of this experiment suggest a coupling between heme synthesis and SDH synthesis. The bacteria were not washed before shifting to medium lacking 5-aminolevulinic acid in this experiment to minimize the growth lag after the shift. Thus, a small amount of growth factor was carried into the new medium. The results of this IC, If 1 J i 1 oc E E O E HEME AND SDH IN B. SUBTILIS 379 experiment and the one shown in Fig. 1 indicate that the bacteria had an excess of cytochrome since they grew at undiminished rate for more than 2 h after the shift. In the following experiment SDH synthesis was followed under more stringent conditions of 5-aminolevulinic acid starvation. An additional purpose of the experiment was to study the effect of readdition of 5-aminolevulinic acid, after a period of starvation for the growth factor, on synthesis of SDH. Strain KAll was grown and shifted to medium without 5-aminolevulinic acid. After 2 h of incubation without growth factor, the bacteria were diluted into medium with or without 5-aminolevulinic acid, and the bacteria were grown for an additional 2 h in these media. Samples were taken at various times, and the amount of SDH antigen was deterinined in whole-cell lysates, in a soluble fraction, and in the membrane fraction. A fixed volume from each sample was run in rocket immunoelectrophoresis against SDH-specific antiserum. The gels were stained for SDH and then for protein with Coomassie brilliant blue. In rocket immunoelectrophoresis the peak area of the precipitate is proportional to the amount of antigen at constant antiserum concentration (2). The relative amount of SDH antigen in each sample was then determined by projecting the precipitates on paper and cutting out and weighing the projected precipitates. The results of these measurements are summarized in Fig. 3, which also shows the growth curves for the cultures. During the first period of 5-aminolevulinic acid starvation, total SDH antigen increases at the same rate as bacterial mass. In this culture there is accumulation of antigen in the cytoplasm, whereas the amount of membrane-bound antigen only increases about twofold as compared to about a fivefold increase in bacterial mass. The reduced incorporation of SDH into membrane is in agreement with the results shown in Fig. 2. On the second shift to medium lacking 5-aminolevulinic acid, net synthesis of SDH antigen is much reduced, and there is no further increase in membrane-bound antigen. In the bacteria shifted to medium containing 5-ammnolevulinic acid, total SDH antigen increased at a faster rate than bacterial mass. There is a rapid initial decrease in cytoplasmic SDH antigen and an increase in membranebound antigen at a rate which is about three times the rate of increase of total SDH antigen. The experiment shown in Fig. 3 was repeated with the following modification. After 2 h of incubation without 5-aminolevulinic acid, the bacteria were shifted to medium containing the growth factor, and samples were taken at 1-min intervals during the first 3 min after the shift.

4 38 HOLMGREN, HEDERSTEDT, AND RUTBERG J. BACTERIOL. 4D c a, Ua# a-. U c CD a._ U I Time (hours) Time (hours) FIG. 3. Strain KAll was grown and shifted to medium without 5-aminolevulinic acid as described in the legend to Fig. 2. Samples were taken every 3 min during a 2-h period. The remaining bacteria were then centrifuged, suspended in medium without 5-aminolevulinic acid, and used to inoculate two new cultures, one with and one without 5-aminolevulinic acid. These cultures were incubated at 37 C with shaking, and samples were taken every 3 min during a 15-min period. The bacteria were lysed as described in the legend to Fig. 1. A portion from each lysate was removed from each sample before membrane preparation and stored at -7 C until used (cell lysate). The top two-thirds of the first supernatant obtained during membrane preparation was recovered and centrifuged at 48,2(X x g for 6 min at 4 C. The resulting supernatant was kept at -7 C until used (cytoplasmic fraction). Membranes were prepared as described (15). The various fractions were assayed for SDH enzymatic activity and for the presence of SDH antigen in rocket immunoelectrophoresis. (A) Growth of bacteria (, without 5-aminokvulinic acid;, with 5-aminolevulinic acid); the arrow indicates the time of the second shift; (B) SDH antigen in total lysate; (C) SDH antigen in cytoplasmic fraction; (D) SDH antigen in membrane. The amount of SDH antigen in the whole-cell lysate, in the membrane fraction, and in the soluble fraction, respectively, was then measured in each sample. The results of this experiment (Fig. 4) confirm that membrane-bound SDH antigen appears at a faster rate after heme synthesis is resumed than can be accounted for by SDH net synthesis. The results of the above experiments thus suggest that cytoplasmic SDH, synthesized during 5-aminolevulinic acid starvation, becomes membrane bound when the bacteria start synthesizing cytochrome. SDH enzyme activity was also measured in each sample. No activity was found in any cytoplasmic fraction, whereas the activity in the membranes was roughly proportional to the amount of SDH antigen detected in immunoelectrophoresis (data not shown). The results with the cytoplasmic fraction and whole lysates CL a, a, cannot be reliably quantitated. One complicating factor is most likely the presence of enzyme inhibitors in these fractions. This makes it important to characterize the soluble antigen which accumulates in 5-aminolevulinic acidstarved bacteria in relation to the membranebound SDH complex. Pattern of total membrane proteins synthesized during 5-aminolevulinic acid starvation of strain KAll. The following experiment was done to examine the effect of 5-aminolevulinic acid starvation on the synthesis of the bulk of membrane proteins in KAll. The bacteria were grown in the presence of 5-aminolevulinic acid and then shifted to medium with and without the growth factor and containing a mixture of "C-labeled L-amino acids. Samples were taken at intervals from the two cultures during a 3-h period, and membranes were

5 VOL. 138, 1979 z w 2. HEME AND SDH IN B. SUBTILIS hours KA z LJ 1.4L U. w ~-xruriffiirrr#ip *'^ ''ZoP-w... w~~~~~~~. w...t. ; z -J TIME (MINUTES) FIG. 4. Amount of SDH antigen in whole-cell lysate (), membrane (U), and soluble (A) fraction after shift of strain KAl1 from medium without 5- aminolevulinic acid to medium with the growth factor. The experiment was performed as described in the legend to Fig. 3, but samples were taken with closer intervals and only during the first 35 min after shift to medium containing 5-aminolevulinic acid. prepared. The membrane proteins were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels,were stained for protein with Coomassie brilliant blue and then autoradiographed. Neither the stained gel nor the autoradiograph reveals any major differences in the polypeptide pattern of membranes from bacteria grown with or without 5-aminolevulinic acid, respectively. However, the gels clearly show that the amount of the 65,-dalton subunit of the SDH complex (9) is much reduced in membranes from 5-aminolevulinic acid-starved bacteria (Fig. 5). The same is true also for the 28,-dalton subunit. We cannot decide from these experiments whether the 19,-dalton subunit, which in the gels is believed to correspond to the apoprotein of cytochrome b (9), is reduced in amount. DISCUSSION In B. subtilis and several other bacterial species, SDH is thought to transfer reducing equiv- polyacrylamide gel electrophoresis of membranes from KA411 grown with (+) and without (-) 5-aminolevulinic acid in a mixture of '4C-labeled L -amino acids. Samples were taken at.5-h intervals during a 3-h period. Amount of protein and radioactivity (counts per minute) loaded on the gel:.5 h, 3 pg, and 6x 13 cpmn; 1 h, 6pg, and 15 x 13 CPM; 1.5 h, 12 pg. and 24 X 13 cpm; 2 h, 12 jg, and 3 x 1 cpm; 2.5 h, 15 jg, and 36 x 13 cpm; 3 h, 18pg, and 42 x1 cpm. The gradient of the gel was 1 to 15% (wt/vol) acrylamide-.26.4% (wt/vol) bisacrylamide. alents to cytochrome b (5, 6, 12). One of the three polypeptides identified in the solubilized SDH complex from B. subtilis is suggested to be a cytochrome b (9). Thus, a close structural and functional coupling between SDH and cytochrome is implied in these bacteria. SDH has been purified in a soluble form from Rhodospirillum rubrum chromatophores (4) and from beef heart mitochondria (3). In both cases the enzyme has two subunits with no cytochrome associated. In the present experiments, we have used a 5-aminolevulinic acid requiring mutant of B. subtilis to examine the role of cytochrolme synthesis on the synthesis and membrane association of SDH. Such mutants have been used for similar purposes in other organisms, e.g.,min Escherichia coli it has been shown that the amount of cytoplasmic nitrate reductase increases when heme synthesis is blocked ( 1), and in bakers' yeast cytochrome c oxidase requires heme synthesis for proper assembly (16). To quantitate the amount of SDH in various samples we have used rocket immunoelectro-

6 382 HOLMGREN, HEDERSTEDT, AND RUTBERG phoresis with an SDH-specific antiserum. This antiserum contains antibody which reacts with the largest, 65,-molecular-weight flavin-containing subunit of the SDH complex (9). We do not know whether it also contains antibody against the other two subunits. In wild-type B. subtilis most of the SDH enzyme activity is found in the membrane fraction (15), indicating a close coupling between synthesis and membrane binding of the enzyme. When the mutant KAll is starved for 5-aminolevulinic acid, the relative amount of membranebound SDH decreases. Slab gels of membrane proteins from starved cells indicate that both the 65,- and the 28,-molecular-weight subunits of the SDH complex are present in decreased amounts in these membranes. During the first 2 h of starvation, increasing amounts of SDH antigen accumulate in the cytoplasm. When heme synthesis is resumed, the amount of cytoplasmic SDH antigen rapidly decreases, whereas the amount of membrane-bound SDH antigen increases at a rate too high to be accounted for only by de novo synthesis of SDH. On the basis of the present results, the following model for synthesis and membrane binding of SDH in B. subtilis can be proposed. The 65,-dalton and the 28,-dalton subunits of the SDH complex are synthesized and associate in the cytoplasm and then bind to a specific site in the membrane. Cytochrome b is an essential part of this binding site, and the membrane binding occurs via an interaction between the 28,-dalton subunit and cytochrome b (7, 9). When the number of membrane sites becomes limiting, SDH synthesis does not stop, but increasing amounts of enzyme accumulate in the cytoplasm. When new membrane sites become available, the cytoplasmic enzyme will bind to these. Reconstitution experiments with isolated, soluble SDH from beef heart mitochondria have shown limiting and defined amounts of SDH binding sites. Alkali-inactivated electron transport particles and complex II from beef heart mitochondria bind one equivalent of soluble, active SDH per membrane-bound, alkali-inactivated SDH (8). The above model is consistent with the present results, with our present knowledge of the composition of the B. subtilis SDH complex, and with the properties of some citf mutants as J. BACTrERIOL. recently described (9, 15). The model also poses several problems which can be studied with presently available techniques, e.g., what are the properties of the soluble SDH antigen which accumulates in the absence of heme synthesis? Is cytochromal aproprotein synthesized and incorporated in the membrane in the absence of heme synthesis? Are the SDH membrane sites selected at random? These problems are currently being studied in our laboratory. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Medical Research Council and from Karolinska Institutet. The expert technical assistance of Kerstin Bernholm and Sven-Ake Franzen is gratefully acknowledged. LITERATURE CITED 1. Anderson, T. J., and G. Ivanovics Isolation and some characteristics of haemin dependent mutants of Bacillus subtilis. J. Gen. Microbiol. 49: Bjerrum,. J Immunochemical investigation of membrane proteins. A methodological survey with emphasis placed on immunoprecipitation in gels. Biochim. Biophys. Acta 472: Davis, K. A., and Y. Hatefi Succinate dehydrogenase. I Purification, molecular properties and substructure. Biochemistry 1: Davis, K. A., Y. Hatefi, I. P. Crawford, and H. Baltscheffsky Purification, Molecular properties and amino acid composition of Rhodospirillum rubrum succinate dehydrogenase. Arch. Biochem. Biophys. 18: Farrand, S. K., and H. W. Taber Physiological effects of menaquinone deficiency in Bacillus subtilis. J. 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