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Presence and stoichiometry of two forms of subunit 6 of the mitochondrial ATPase complex of yeast
ARCHIVES OF BIOCHEMISTRY Vol. 227, No. 1, November,
AND
BIOPHYSICS
pp. 106-110, 1983
Presence and Stoichiometry of Two Forms of Subunit 6 of the Mitochondrial ATPase Complex of Yeast’ RICHARD D. TODD’ Department
of Biochemistry,
University
Received March
MICHAEL
AND
of Texas Health
G. DOUGLAS3
Science &r&r,
San Antonio,
Texas 78284
11, 1983, and in revised form June 23, 1983
One of the mitochondrically coded components of the yeast mitochondrial ATPase complex (subunit 6) can be resolved into two components on certain polyacrylamide gels in the presence of sodium dodecyl sulfate. Purification of the ATPase complex from commercially processed yeast as well as immunoprecipitation of the holo-enzyme from cells labeled in vivo with 14C-labeled amino acids demonstrate that both forms of subunit 6 are physically associated with the assembled enzyme and present in two copies each per complex. One-dimensional papain-generated peptide maps of the two components are identical except for the mobility of a single fragment. It is concluded that the two components of subunit 6 are different forms of a single protein and are present on an average of two copies each per complex.
The molecular events involved in the assembly of a membrane-bound heterooligomer like the mitochondrial ATPase complex as well as its function require additional analysis of structure. The oligomycin-sensitive complex of yeast contains at least nine nonidentical protein components of dual genetic origin (1). Subunit 6 is a mitochondrially coded and synthesized component of the Fo4 portion of the yeast mitochondrial ATPase complex (l-2). Under certain conditions subunit 6 can be resolved into two components, termed subunits 6a and 6b (3, 4), which are both under the genetic control of the
Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Unlabeled mitochondrial ATPase complex was isolated from commercially prepared yeast Succharo myces cerertiiae (Red Star Corp.) by either the Triton (7) or the Triton/deoxycholate methods (8) as described previously. Radioactively labeled ATPase complex was isolated by the Triton method from laboratory strain D273-10B ATCC (24567;otPET p’) grown 14 generations in the presence of a “C-labeled amino acid mixture, as previously described (5). Cytochiome oxidase was the kind gift of Michael E. Dotter, St. Judes Children’s Research Hospital, Memphis, Tennessee. Immunoprecipitations, SDS-gel electrophoresis of ATPase complex, autoradiography, Coomassie blue
’ This investigation was supported by a grant from the National Institutes of Health (GM 26713) and Grant AQ-814 from the Robert A. Welch Foundation. * Present address: Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, Calif. 94305. ‘To whom requests for reprints should be addressed. ’ Abbreviations used: F,-ATPase, the membraneassociated portion of the ATPase complex; F,-ATPase, the water-soluble portion of the ATPase complex (coupling factor 1); SDS, sodium dodecyl sulfate. 0003-9861/83 $3.00
oli 2 locus of the yeast mitochondrial genome (4). The total number of copies of subunit 6 per ATPase complex is four (5). Chemical crosslinking studies of the solubilized ATPase complex have shown that subunit 6 is located near itself and subunits 1, 4, and 9 (2, 6). In the present study we show that the two forms of subunit 6 are present in two copies each per ATPase complex and differ in a single papain-generated peptide fragment.
106
YEAST
MITOCHONDRIAL
SUBUNIT
107
6
staining, and densinometric scanning of gels were all performed exactly as previously described (5). One-dimensional peptide maps were performed as described by Cleveland et al. (9) except that the proteins were first modified with ‘%I by the Iodogen method as previously described (6). Proteins were cut out of Coomassie blue-stained SDS-acrylamide slab gels. The gel slices were soaked for 30 min in sample buffer (5) and 1% SDS and then transferred to Iodogen-coated tubes. An aliquot of 0.1 to 0.5 mCi of ‘%I was added per gel slice and the tubes were gently agitated for 2 h. The reaction was stopped by removing the izI solution and the gel slices were soaked for 2 h in 10% methanol to remove unreacted iodine. The gel slices were soaked for 30 min in sample buffer and then placed in the wells of a 15 or 17% acrylamide:Ol% SDS slab gel for proteolysis and electrophoresis. Samples without added papain electrophoresed as single species indicating little cross-contamination from other proteins when gel slices were cut and little proteolysis or breakdown during labeling and processing. The materials used were W-labeled amino acid mixture (algal protein hydrolysate, 0.2 to 0.4 Ci/mmol, ICN); ‘=I, carrier free (ICN); papain (Sigma), Iodogen (Pierce Chemical).
I 2
3 4 5 6
ATPase
ab
7 8 9
RESULTS
FIG. 1. Presence of two forms of subunit 6 on an SDS-acrylamide gel. Triton-solubilized ATPase complex (30 fig) was electrophoresed on a lo-17.5% acrylamide:O.l% SDS slab gel and stained with Coomassie blue. Numbers identify the subunits. The direction of electrophoresis was from top to bottom.
AND
DISCUSSION
Subunit 6 of the ATPase complex is inconsistently resolved into two components by SDS-gel electrophoresis (4) (Fig. 1). The basis for this inconsistent separation is unknown, but is not a function of storage, proteolysis, or particular ATPase preparation since subunit 6 from the same preparation of solubilized ATPase complex has
*
Electrophoresis FIG. 2. Immunoprecipitation of both forms of subunit 6 with the ATPase complex. Mitochondria of yeast grown for 14 generations in the presence of W-labeled amino acids were sonicated and solubilized with 1% Triton as described previously (5) and then incubated with rabbit antiserum directed against yeast mitochondrial F,-ATPase. The immunoprecipitates were processed as described previously (5) and electrophoresed on a lo-17.4% acrylamide:O.l% SDS slab gel. The gel was dried and autoradiographed, and the autoradiogram was scanned as before (5). Under the conditions used, the area under each peak is directly proportional to the radioactivity present in each protein.
108
TODD
AND TABLE
DOUGLAS I
STOICHIOMETRY OF SUBUNITS 6a AND 6b
Subunit
Molecular weight (X10-y
3 6a 6b
32.0 21.9 21.9
Relative amounts of labelb 1.0 1.28 1.36
Observed stoichiometry” 1.0’ 1.87 1.99
Assumed stoichiometryd 1.0 2.0 2.0
“From Ref. (5). b Determined by normalizing the areas under the curves in Fig. 2 to the area of the subunit 3 curve. ‘Derived by dividing the relative amount of label for each subunit by its molecular weight and normalizing all values to subunit 3. d Derived by rounding the observed stoichiometries to the nearest integer. “The assumed stoichiometry of one copy of subunit 3 is based on our previously reported subunit stoichiometries for the entire ATPase complex (5).
ATPase
CO
I II In m;‘;T m YII FIG. 3. Comparison of the protein compositions of the mitochondrial ATPase complex and the cytochrome oxidase complex. Samples of each complex (30 pg) were electrophoresed on a lo-17.5% acrylamide:O.l% SDS slab gel and stained with Coomassie blue. The ATPase subunits are identified by arabic numerals, the cytochrome oxidase subunits by roman numerals. Cytochrome oxidase (CO) electrophoresis was from top to bottom. The ATPase complex was prepared by the Triton/deoxycholate method (8) and therefore has 11 subunits.
been resolved into one, then two, and then one component on successive gels (not shown). Nor is the separation detergent specific since subunit 6 of both Triton (‘7)and Triton/deoxycholate (8)-solubilized complex may be resolved into one or two components. Small differences between gels, such as variations in detergent concentration or acrylamide concentration or both, seem the most likely explanation. Immunoprecipitation analysis indicates that both components of subunit 6 are firmly associated with the complex. As shown in Fig. 2, immunoprecipitation with antisera directed against the F,-ATPase coprecipitates both components of subunit 6. Further, the immunoprecipitation of ATPase complex grown in the presence of 14C-labeled amino acids allows the calculation of the stoichiometry of the two forms of subunit 6 (5). As indicated in Table I, this analysis indicates that both subunits 6a and 6b are present in two copies per ATPase complex. Whether some complexes contain only 6a or 6b or whether any mixture of 6a and 6b is possible in an individual complex is not known. As previously noted by others (4), subunit III of the yeast mitochondrial cytochrome oxidase complex has an electrophoretic mobility similar to subunit 6 of the ATPase complex. As shown in Fig. 3 cytochrome oxidase subunits II, III, and IV comigrate with ATPase complex subunits
YEAST
MITOCHONDRIAL
ATPase
SUBUNIT
6
FIG. 4. (a) Comparison of the papain-generated fragments of ATPase subunits 6a and 6b and cytochrome oxidase subunit 3 (CO III). The individual subunits were cut out of slab gels (such as in Figs. 1 and 3) and labeled with ‘%I as described under Materials and Methods, and the gel slices were put in the wells of a 15% acrylamide:O.l% SDS slab gel. The slices were then covered with sample buffer containing 20% glycerol and 0.5 pg of papain per well. Samples were electrophoresed until the bromphenol blue dye approached the end of the separating gel. The current was turned off for 30 min (to allow proteolysis) and then electrophoresis was completed. The gels were dried, autoradiogramed, and scanned as before (5). The roman numerals (I, II, and III) identify the major papain fragments of subunits 6a and b. The papain-derived pattern of subunit 6b is compared to subunit 6a plus 6b. All manipulations are the same except a 17% acrylamide: 0.1% SDS gel was used for better resolution of papain fragments I and II. Only the fragment I and II portion of the scans are shown.
3, 6, and 7, respectively. In order to determine the relationship of ATPase subunit 6a to 6b and to cytochrome oxidase subunit III, one-dimensional peptide maps (9) were performed on the respective subunits using papain. Figure 4a shows densitometric scans of autoracliograms of 1251-labeled papain fragments of subunits 6a, 6b, and nonresolved subunit 6 (6a + 6b) of the ATPase complex in addition to cytochrome oxiclase subunit III. The peptide fragments were separated on a 15% acrylamide: 0.1% SDS slab gel. The papain-generated fragment pattern of cytochrome oxidase subunit III is completely different from the ATPase complex subunit 6 patterns. This is consistent with the previously reported differences in isoelectric points for cytochrome oxidase subunit III and the two forms of ATPase subunit 6 (4). The subunit 6 patterns resemble each other. The only reproducible difference between the patterns of 6a and 6b is the shift in mobility of a single major fragment (labeled II in Fig. 4a). This difference is shown
in more detail on a higher percentage acrylamide gel in Fig. 4b. We conclude that subunits 6a and 6b differ in at least a single ‘%I-labeled papain-generated fragment and that both are different from cytochrome oxiclase subunit III. Recently, Stephenson et al. suggested that the two forms of subunit 6 of the ATPase complex (and the two forms of cytochrome oxidase subunit II, both of yeast mitochondria) might represent either posttranslational modifications of single species, particularly a precursor product relationship, or different proteins coded by different genes (4). These earlier studies observed that the relative amount of subunit 6a and b detected in mitochonclrial membranes was not equal following in vivo labeling of mitochondrial-specific products (10). However, since both forms of subunit 6 are present in equimolar amounts in assembled ATPase complex under steadystate conditions (cells grown 14 generations in 14C-labeled amino acids; Fig. 2 and Table I) it is unlikely that one form represents
110
TODD
AND
a precursor protein which is modified. The papain-fragment patterns of subunits 6a and 6b are nearly identical, indicating that it is unlikely that they represent dissimilar or functionally distinct proteins which are both under the control of the oli 2 locus (4, 10). This possibility is considered most unlikely in light of the DNA sequence of this region (11) and the observation that single mitochondrial oli 2 mit- mutations alter both forms of the subunit (10). Whatever the difference is between subunit 6a and 6b, it appears to be localized to the papain-generated fragment II. Whether subunit 6a or 6b or both represent the protein product predicted from DNA sequencing of the oli 2 region (11) remains to be firmly established. ACKNOWLEDGMENTS The expert technical assistance of M. Buck and T. Griesenbeck in this study is greatly acknowledged. REFERENCES 1. TZAGOLOFF, A., AND MEAGHER, P. (1972) J. Biol. Chem. 247, 594-603.
DOUGLAS 2. ENNS, R., AND CRIDDLE, R. S. (1977) Arch Biochem Biophys. 183, 742-752. 3. TODD, R., GRIESENBECK, T., MCADA, P., BUCK, M., AND DOUGLAS, M. (1980) in The Organization and Expression of the Mitochondrial Genome (Kroon, A. M., and Saccone, C., eds.), pp. 375381, Elsevier/North-Holland, Amsterdam/New York. 4. STEPHENSON, G., MARZUKI, S., AND LINNANE, A. W. (1980) Biachem Bivphys. Actu 609,329341. 5. TODD, R. D., GRIESENBECK, T., AND DOUGLAS, M. G. (1980) J. Biol. Chem 255,5461-5467. 6. TODD, R. D., AND DOUGLAS, M. G. (1981) J. Biol Chem 256, 6984-6989. 7. DOUGLAS, M. G., KOH, Y., EBNER, E., AGSTERIBBE, E., AND SCHATZ, G. (1979) J. Biol. Chem 254, 1335-1339. 8. RYRIE, I. J., AND GALLAGHER, A. (1979) Biochem Biophys. Acta 545, 1-14. 9. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M. W., AND LAEMMLI, U. K. (1977) J. Bid Chem 252, 1102-1106. 10. STEPHENSON, G., MARZUKI, S., AND LINNANE, A. W. (1981) B&hem. Biophys. Actu 636, 104112. 11. MACINO, G., AND TZAGOLOFF, A. (1980) Cell 20, 507-517.
AND
BIOPHYSICS
pp. 106-110, 1983
Presence and Stoichiometry of Two Forms of Subunit 6 of the Mitochondrial ATPase Complex of Yeast’ RICHARD D. TODD’ Department
of Biochemistry,
University
Received March
MICHAEL
AND
of Texas Health
G. DOUGLAS3
Science &r&r,
San Antonio,
Texas 78284
11, 1983, and in revised form June 23, 1983
One of the mitochondrically coded components of the yeast mitochondrial ATPase complex (subunit 6) can be resolved into two components on certain polyacrylamide gels in the presence of sodium dodecyl sulfate. Purification of the ATPase complex from commercially processed yeast as well as immunoprecipitation of the holo-enzyme from cells labeled in vivo with 14C-labeled amino acids demonstrate that both forms of subunit 6 are physically associated with the assembled enzyme and present in two copies each per complex. One-dimensional papain-generated peptide maps of the two components are identical except for the mobility of a single fragment. It is concluded that the two components of subunit 6 are different forms of a single protein and are present on an average of two copies each per complex.
The molecular events involved in the assembly of a membrane-bound heterooligomer like the mitochondrial ATPase complex as well as its function require additional analysis of structure. The oligomycin-sensitive complex of yeast contains at least nine nonidentical protein components of dual genetic origin (1). Subunit 6 is a mitochondrially coded and synthesized component of the Fo4 portion of the yeast mitochondrial ATPase complex (l-2). Under certain conditions subunit 6 can be resolved into two components, termed subunits 6a and 6b (3, 4), which are both under the genetic control of the
Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Unlabeled mitochondrial ATPase complex was isolated from commercially prepared yeast Succharo myces cerertiiae (Red Star Corp.) by either the Triton (7) or the Triton/deoxycholate methods (8) as described previously. Radioactively labeled ATPase complex was isolated by the Triton method from laboratory strain D273-10B ATCC (24567;otPET p’) grown 14 generations in the presence of a “C-labeled amino acid mixture, as previously described (5). Cytochiome oxidase was the kind gift of Michael E. Dotter, St. Judes Children’s Research Hospital, Memphis, Tennessee. Immunoprecipitations, SDS-gel electrophoresis of ATPase complex, autoradiography, Coomassie blue
’ This investigation was supported by a grant from the National Institutes of Health (GM 26713) and Grant AQ-814 from the Robert A. Welch Foundation. * Present address: Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, Calif. 94305. ‘To whom requests for reprints should be addressed. ’ Abbreviations used: F,-ATPase, the membraneassociated portion of the ATPase complex; F,-ATPase, the water-soluble portion of the ATPase complex (coupling factor 1); SDS, sodium dodecyl sulfate. 0003-9861/83 $3.00
oli 2 locus of the yeast mitochondrial genome (4). The total number of copies of subunit 6 per ATPase complex is four (5). Chemical crosslinking studies of the solubilized ATPase complex have shown that subunit 6 is located near itself and subunits 1, 4, and 9 (2, 6). In the present study we show that the two forms of subunit 6 are present in two copies each per ATPase complex and differ in a single papain-generated peptide fragment.
106
YEAST
MITOCHONDRIAL
SUBUNIT
107
6
staining, and densinometric scanning of gels were all performed exactly as previously described (5). One-dimensional peptide maps were performed as described by Cleveland et al. (9) except that the proteins were first modified with ‘%I by the Iodogen method as previously described (6). Proteins were cut out of Coomassie blue-stained SDS-acrylamide slab gels. The gel slices were soaked for 30 min in sample buffer (5) and 1% SDS and then transferred to Iodogen-coated tubes. An aliquot of 0.1 to 0.5 mCi of ‘%I was added per gel slice and the tubes were gently agitated for 2 h. The reaction was stopped by removing the izI solution and the gel slices were soaked for 2 h in 10% methanol to remove unreacted iodine. The gel slices were soaked for 30 min in sample buffer and then placed in the wells of a 15 or 17% acrylamide:Ol% SDS slab gel for proteolysis and electrophoresis. Samples without added papain electrophoresed as single species indicating little cross-contamination from other proteins when gel slices were cut and little proteolysis or breakdown during labeling and processing. The materials used were W-labeled amino acid mixture (algal protein hydrolysate, 0.2 to 0.4 Ci/mmol, ICN); ‘=I, carrier free (ICN); papain (Sigma), Iodogen (Pierce Chemical).
I 2
3 4 5 6
ATPase
ab
7 8 9
RESULTS
FIG. 1. Presence of two forms of subunit 6 on an SDS-acrylamide gel. Triton-solubilized ATPase complex (30 fig) was electrophoresed on a lo-17.5% acrylamide:O.l% SDS slab gel and stained with Coomassie blue. Numbers identify the subunits. The direction of electrophoresis was from top to bottom.
AND
DISCUSSION
Subunit 6 of the ATPase complex is inconsistently resolved into two components by SDS-gel electrophoresis (4) (Fig. 1). The basis for this inconsistent separation is unknown, but is not a function of storage, proteolysis, or particular ATPase preparation since subunit 6 from the same preparation of solubilized ATPase complex has
*
Electrophoresis FIG. 2. Immunoprecipitation of both forms of subunit 6 with the ATPase complex. Mitochondria of yeast grown for 14 generations in the presence of W-labeled amino acids were sonicated and solubilized with 1% Triton as described previously (5) and then incubated with rabbit antiserum directed against yeast mitochondrial F,-ATPase. The immunoprecipitates were processed as described previously (5) and electrophoresed on a lo-17.4% acrylamide:O.l% SDS slab gel. The gel was dried and autoradiographed, and the autoradiogram was scanned as before (5). Under the conditions used, the area under each peak is directly proportional to the radioactivity present in each protein.
108
TODD
AND TABLE
DOUGLAS I
STOICHIOMETRY OF SUBUNITS 6a AND 6b
Subunit
Molecular weight (X10-y
3 6a 6b
32.0 21.9 21.9
Relative amounts of labelb 1.0 1.28 1.36
Observed stoichiometry” 1.0’ 1.87 1.99
Assumed stoichiometryd 1.0 2.0 2.0
“From Ref. (5). b Determined by normalizing the areas under the curves in Fig. 2 to the area of the subunit 3 curve. ‘Derived by dividing the relative amount of label for each subunit by its molecular weight and normalizing all values to subunit 3. d Derived by rounding the observed stoichiometries to the nearest integer. “The assumed stoichiometry of one copy of subunit 3 is based on our previously reported subunit stoichiometries for the entire ATPase complex (5).
ATPase
CO
I II In m;‘;T m YII FIG. 3. Comparison of the protein compositions of the mitochondrial ATPase complex and the cytochrome oxidase complex. Samples of each complex (30 pg) were electrophoresed on a lo-17.5% acrylamide:O.l% SDS slab gel and stained with Coomassie blue. The ATPase subunits are identified by arabic numerals, the cytochrome oxidase subunits by roman numerals. Cytochrome oxidase (CO) electrophoresis was from top to bottom. The ATPase complex was prepared by the Triton/deoxycholate method (8) and therefore has 11 subunits.
been resolved into one, then two, and then one component on successive gels (not shown). Nor is the separation detergent specific since subunit 6 of both Triton (‘7)and Triton/deoxycholate (8)-solubilized complex may be resolved into one or two components. Small differences between gels, such as variations in detergent concentration or acrylamide concentration or both, seem the most likely explanation. Immunoprecipitation analysis indicates that both components of subunit 6 are firmly associated with the complex. As shown in Fig. 2, immunoprecipitation with antisera directed against the F,-ATPase coprecipitates both components of subunit 6. Further, the immunoprecipitation of ATPase complex grown in the presence of 14C-labeled amino acids allows the calculation of the stoichiometry of the two forms of subunit 6 (5). As indicated in Table I, this analysis indicates that both subunits 6a and 6b are present in two copies per ATPase complex. Whether some complexes contain only 6a or 6b or whether any mixture of 6a and 6b is possible in an individual complex is not known. As previously noted by others (4), subunit III of the yeast mitochondrial cytochrome oxidase complex has an electrophoretic mobility similar to subunit 6 of the ATPase complex. As shown in Fig. 3 cytochrome oxidase subunits II, III, and IV comigrate with ATPase complex subunits
YEAST
MITOCHONDRIAL
ATPase
SUBUNIT
6
FIG. 4. (a) Comparison of the papain-generated fragments of ATPase subunits 6a and 6b and cytochrome oxidase subunit 3 (CO III). The individual subunits were cut out of slab gels (such as in Figs. 1 and 3) and labeled with ‘%I as described under Materials and Methods, and the gel slices were put in the wells of a 15% acrylamide:O.l% SDS slab gel. The slices were then covered with sample buffer containing 20% glycerol and 0.5 pg of papain per well. Samples were electrophoresed until the bromphenol blue dye approached the end of the separating gel. The current was turned off for 30 min (to allow proteolysis) and then electrophoresis was completed. The gels were dried, autoradiogramed, and scanned as before (5). The roman numerals (I, II, and III) identify the major papain fragments of subunits 6a and b. The papain-derived pattern of subunit 6b is compared to subunit 6a plus 6b. All manipulations are the same except a 17% acrylamide: 0.1% SDS gel was used for better resolution of papain fragments I and II. Only the fragment I and II portion of the scans are shown.
3, 6, and 7, respectively. In order to determine the relationship of ATPase subunit 6a to 6b and to cytochrome oxidase subunit III, one-dimensional peptide maps (9) were performed on the respective subunits using papain. Figure 4a shows densitometric scans of autoracliograms of 1251-labeled papain fragments of subunits 6a, 6b, and nonresolved subunit 6 (6a + 6b) of the ATPase complex in addition to cytochrome oxiclase subunit III. The peptide fragments were separated on a 15% acrylamide: 0.1% SDS slab gel. The papain-generated fragment pattern of cytochrome oxidase subunit III is completely different from the ATPase complex subunit 6 patterns. This is consistent with the previously reported differences in isoelectric points for cytochrome oxidase subunit III and the two forms of ATPase subunit 6 (4). The subunit 6 patterns resemble each other. The only reproducible difference between the patterns of 6a and 6b is the shift in mobility of a single major fragment (labeled II in Fig. 4a). This difference is shown
in more detail on a higher percentage acrylamide gel in Fig. 4b. We conclude that subunits 6a and 6b differ in at least a single ‘%I-labeled papain-generated fragment and that both are different from cytochrome oxiclase subunit III. Recently, Stephenson et al. suggested that the two forms of subunit 6 of the ATPase complex (and the two forms of cytochrome oxidase subunit II, both of yeast mitochondria) might represent either posttranslational modifications of single species, particularly a precursor product relationship, or different proteins coded by different genes (4). These earlier studies observed that the relative amount of subunit 6a and b detected in mitochonclrial membranes was not equal following in vivo labeling of mitochondrial-specific products (10). However, since both forms of subunit 6 are present in equimolar amounts in assembled ATPase complex under steadystate conditions (cells grown 14 generations in 14C-labeled amino acids; Fig. 2 and Table I) it is unlikely that one form represents
110
TODD
AND
a precursor protein which is modified. The papain-fragment patterns of subunits 6a and 6b are nearly identical, indicating that it is unlikely that they represent dissimilar or functionally distinct proteins which are both under the control of the oli 2 locus (4, 10). This possibility is considered most unlikely in light of the DNA sequence of this region (11) and the observation that single mitochondrial oli 2 mit- mutations alter both forms of the subunit (10). Whatever the difference is between subunit 6a and 6b, it appears to be localized to the papain-generated fragment II. Whether subunit 6a or 6b or both represent the protein product predicted from DNA sequencing of the oli 2 region (11) remains to be firmly established. ACKNOWLEDGMENTS The expert technical assistance of M. Buck and T. Griesenbeck in this study is greatly acknowledged. REFERENCES 1. TZAGOLOFF, A., AND MEAGHER, P. (1972) J. Biol. Chem. 247, 594-603.
DOUGLAS 2. ENNS, R., AND CRIDDLE, R. S. (1977) Arch Biochem Biophys. 183, 742-752. 3. TODD, R., GRIESENBECK, T., MCADA, P., BUCK, M., AND DOUGLAS, M. (1980) in The Organization and Expression of the Mitochondrial Genome (Kroon, A. M., and Saccone, C., eds.), pp. 375381, Elsevier/North-Holland, Amsterdam/New York. 4. STEPHENSON, G., MARZUKI, S., AND LINNANE, A. W. (1980) Biachem Bivphys. Actu 609,329341. 5. TODD, R. D., GRIESENBECK, T., AND DOUGLAS, M. G. (1980) J. Biol. Chem 255,5461-5467. 6. TODD, R. D., AND DOUGLAS, M. G. (1981) J. Biol Chem 256, 6984-6989. 7. DOUGLAS, M. G., KOH, Y., EBNER, E., AGSTERIBBE, E., AND SCHATZ, G. (1979) J. Biol. Chem 254, 1335-1339. 8. RYRIE, I. J., AND GALLAGHER, A. (1979) Biochem Biophys. Acta 545, 1-14. 9. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M. W., AND LAEMMLI, U. K. (1977) J. Bid Chem 252, 1102-1106. 10. STEPHENSON, G., MARZUKI, S., AND LINNANE, A. W. (1981) B&hem. Biophys. Actu 636, 104112. 11. MACINO, G., AND TZAGOLOFF, A. (1980) Cell 20, 507-517.