Cell, Vol. 10. 721-728,
April
1977, Copyright
0 1977 by MIT
Two Homogeneous Myosins Caenorhabditis elegans Frederick Henry F. Department Stanford Stanford,
H. Schachat, Harriet E. Harris Epstein of Pharmacology University School of Medicine California 94305
and
Summary Myosin purified from the body-wall muscle-defective mutant E675 of the nematode, Caenorhabditis elegans, has heavy chain polypeptides which can be distinguished on the basis of molecular weight. On SDS-polyacrylamide gels, bands are found at 210,000 and 203,000 daltons. This is in contrast to myosin from the wild-type, N2, which has a single heavy chain band at 210,000 daltons. Both heavy chains of E675 are found in body-wall muscle (Epstein, Waterston and Brenner, 1974). When native myosin from E675 is fractionated on hydroxyapatite, it is separated into myosin containing predominantly one or the other molecular weight heavy chain and myosin containing a mixture of the heavy chains. Comparison of the CNBr fragments of myosin that contains predominantly 210,000 dalton heavy chains with those of myosin that contains predominantly 203,000 dalton heavy chains reveals multiple differences. These differences are not explained by the difference in molecular weight of the heavy chains, but may be explained if each type of heavy chain is the product of a different structural gene. Furthermore, because there are fractions which exhibit >60% 210,000 or >60% 203,000 dalton heavy chain, there is myosin which is homogeneous for each of the heavy chains. Although N2 myosin has only a single molecular weight heavy chain, it too is fractionated by hydroxyapatite. By comparing the CNBr fragments of different myosin fractions, we show that N2, like E675, has two kinds of heavy chains. E190, a body-wall muscle-defective mutant in the same complementation group as E675, is lacking the myosin heavy chain affected by the e675 mutation. This property has allowed us to determine by co-purification of labeled El90 myosin in the presence of excess, unlabeled E675 myosin that most, if not all, of the myosin that contains two different molecular weight heavy chains is due to the formation of complexes between homogeneous myosins and not to a heterogeneous myosin. Introduction Starr and Offer (1973) reported that heavy chains of rabbit fast skeletal muscle myosin had two different
in Body-Wall
Muscle of
amino-terminal amino acid sequences. Although alternative hypotheses were considered, they proposed as the most probable explanation that there are at least two structural genes for rabbit fast skeletal muscle myosin heavy chains. Epstein et al. (1974) in their studies on myosin from the body-wall muscle-defective mutant E675 of the nematode, Caenorhabditis elegans, also found evidence for myosin heavy chain multiplicity in a single muscle type. SDS-polyacrylamide gel electrophoresis of actomyosin extracts from E675 revealed two different myosin heavy chains which were assigned masses of 210,000 and 203,000 daltons. In contrast, actomyosin extracts from the wild-type, N2, showed only a single 210,000 dalton heavy chain. Both molecular weight heavy chains of E675 were located in body-wall muscle. As the most straightforward explanation of these observations, they proposed that the heavy chains of bodywall muscle myosin in wild-type C. elegans were specified by two nonallellic structural genes, and that the mutation in E675 resulted in the conversion of one of the 210,000 dalton gene products to 203,000 daltons. In this paper, we confirm this proposal of Epstein et al. (1974). Further, we show that each of the different body-wall muscle myosin heavy chains associates with a like heavy chain in the native myosin molecule, which is dimeric with respect to the heavy chains. Chromatography of purified E675 myosin on hydroxyapatite resolves myosins homogeneous with respect to either the 210,000 or the 203,000 dalton heavy chains. Comparison of the CNBr peptides of these heavy chains suggests that they are the products of different structural genes. Using the CNBr fragments to detect the different heavy chains, we show that both are present in the wild-type and that its myosin fractionates similarly on hydroxyapatite. Analysis of another mutant, E190, in the same gene as E675 shows that it lacks the homogeneous myosin affected by the e675 mutation. This property allowed us to determine, by co-purification of El90 and E675 myosins, whether fractions from the E675 hydroxyapatite chromatography containing myosin with both heavy chains were due to a heterogeneous myosin species (one which has one of each type of heavy chain). The results indicate that most, if not all, of this mixed material is due to complexes between the two homogeneous myosin species. Results The E675 Myosin Heavy Chains Co-purify Figure 1 D shows the two predominant heavy chains of purified E675 myosin which can be distinguished by SDS-polyacrylamide gel electrophoresis. There
Cdl 722
is also a minor band at 206,000 daltons which Epstein et al. (1974) found in both E675 and N2, and which is located in pharyngeal muscle. Because of its molecular weight, the 206,000 dalton species was putatively identified as a myosin heavy chain. Figure l A-l D show the heavy chain composition of E675 myosin at various stages of its purification. The ratio of 203,000 to 210,000 dalton bands as determined by densitometry of 35S-labeled polypeptides remains constant, within experimental error, during the purification, varying from 1.91 in the homogenate to 2.12 in the Sepharose-purified myosin with a mean and standard deviation of 1.96 t 0.05. The 206,000 dalton band varies between 7% and 10% of the total of the three bands. The ratio of the two predominant heavy chain bands varies from preparation to preparation, but the 203,000 dalton heavy chains is always the major species. The 203,000 to 210,000 dalton ratio was 1.77 ? 0.17 in five homogenates analyzed by densitometry of Coomassie brilliant blue stain or of 35Sautoradiographs. Separation of Myosins Homogeneous for the 203,000 and 210,000 Dalton Heavy Chains When native E675 myosin is chromatographed on hydroxyapatite, species homogeneous for the 203,000 and 210,000 dalton heavy chains are resolved. Figure 2A shows the chromatogram when 600 pg of Sepharose-purified E675 myosin are eluted with a linear phosphate gradient from 0.005 M to 0.5 M with constant 0.6 M KCI, 0.001 M dithiothreitol, 0.0005 M phenylmethylsulfonylfluoride and 0.1% Triton X-100 at a constant pH of 6.5. The profile is complex with three apparent regions: a leading shoulder, fractions 70-89 (region I); a central peak, fractions 92-96 (region II); and a trailing shoulder, fractions 98-110 (region Ill). Since the different molecular weight heavy chains are clearly resolved by SDS-polyacrylamide gel electrophoresis, we are able to quantitate the separation of the two chains by densitometry. Inspection of Figure 3, an autoradiograph of the SDS-polyacrylamide gels on the ?S-myosin from the hydroxyapatite fractions, reveals that region I contains myosin which has predominantly 210,000 dalton heavy chain and region III myosin containing predominantly 203,000 dalton heavy chain. Fractions 75-80 contain myosin that has >80% 210,000 dalton heavy chains, and fractions 100-I 10 contain myosin with >80% 203,000 dalton heavy chains. This demonstrates that native E675 myosin, before electrophoresis under denaturing conditions, contains molecules that are homogeneous for either the 210,000 or 203,000 dalton heavy chains. Figure 26 shows the fractionation of the 210,000 and 203,000 dalton chains separately. The peak fractions are 87 and 103, respectively. Assuming that
A
B
C
D
210 kd
FRONT
Figure
1. Co-purification
of E675 Myosin
Heavy
Chains
Autoradiograph of samples from the purification of Yi-E675 myosin electrophoresed on 4.5% polyacrylamide-SDS gels to show the 210,000 and 203,000 dalton heavy chain polypeptides. (A) the homogenate; (8) the actomyosin extract; (C) the 100,000 supernatant 4; (D) purified myosin. Purification fractions refer to Harris and Epstein (1977).
the separation of chains reflects the separation of three myosin populations-a 210,000 dalton homogeneous, a 203,000 dalton homogeneous and a mixed myosin-we estimate that 24% of the material is homogeneous for the 210,000 dalton heavy chain, 52% is homogeneous for the 203,000 dalton heavy chain and 15% can be represented as myosin containing the two heavy chains. The remaining 8% is 206,000 dalton material and has a peak fraction 74. Its co-purification with myosin and co-migration with myosin on hydroxyapatite lend further support for its identification as a pharyngeal myosin heavy chain. To confirm the identification of two homogeneous myosins, fractions from regions I and III were
Two
Nematode
Body-Wall
Myosins
723
I
14-
I
I
1
1
1
"d'
’
""1
A
12-
8 6 4
T6 -
I
2 0 1011
I ’ -
B
1 ’ ?
’
1 ’ 1
‘D
8642-
ing buffer for the chromatography. Upon rechromatography, this myosin elutes with a maximum at fraction 87 (Figure 2C). When myosin from region III (fractions 101-108) composed of 67% 203,000 dalton heavy chain is similarly treated, it rechromatographs with a maximum at fraction 103 (Figure 2D). These maxima correspond to the maxima of 210,000 and 203,000 dalton myosin heavy chains from the densitometric analysis of the original chromatography (Figure 2B). Thus the homogeneous myosins of E675 rechromatograph true. When myosin containing both molecular weight heavy chains from region II (fractions 92-97) was rechromatographed, it was clear that the 203,000 dalton homogeneous myosin was a major component. The second maximum of the rechromatography at fractions 98-103 corresponds to the elution position of the 203,000 dalton homogeneous myosin. Densitometric analysis of SDS-polyacrylamide gels of the rechromatographed fraction is consistent with this interpretation. However, the absence of 210,000 dalton homogeneous myosin in the rechromatography suggests that the heavy chains do not exchange between myosin molecules during hydroxyapatite chromatography.
ol 14 70
90 FB A:,b?d
10 -
7 ,o x
B 8
8642-
Figure
2. Hydroxyapatite
Chromatography
FRACTION NO. of E675 Myosin
Chromatograms of hydroxyapatite chromatography of %-E675 myosin and rechromatography of fractions of the initial chromatography. Hydroxyapatite columns were run at 2-3 ml/hr. and 1 ml samples were collected. (A) chromatography of E675 myosin: 50 AI samples from each fraction counted as in Experimental Procedures. (6) densitometric analysis of the chromatography based on gels of each of the fractions (Figure 3). (O-O) represents the 210,000 dalton heavy chain; (O-O) the 203,000 dalton heavy chain. Ratios of the chains were determined by densitometry. and the fraction of counts due to each was plotted. (C). (D) and (E) are the rechromatography of fractions 73-81. 92-97 and 101-106, respectively. 100 ~1 samples from each fraction were counted as in Experimental Procedures.
rechromatographed. Myosin from region I (fractions 73-81) composed of 84% 210,000 dalton myosin heavy chain was dialyzed against the start-
Both Homogeneous Myosins Contain 18,000 and 16,000 Dalton Light Chains Harris and Epstein (1977) showed that two light chains of 18,000 and 16,000 daltons are observed in SDS-polyacrylamide gel electrophoresis of N2 myosin. Both these light chains are present in E675 myosin. Figure 4 shows the light chains of Sepharose-purified myosin and those of myosin from the fractions of regions I and Ill which were pooled for rechromatography. The material in region I contains some diffuse material of mass
April
1977, Copyright
0 1977 by MIT
Two Homogeneous Myosins Caenorhabditis elegans Frederick Henry F. Department Stanford Stanford,
H. Schachat, Harriet E. Harris Epstein of Pharmacology University School of Medicine California 94305
and
Summary Myosin purified from the body-wall muscle-defective mutant E675 of the nematode, Caenorhabditis elegans, has heavy chain polypeptides which can be distinguished on the basis of molecular weight. On SDS-polyacrylamide gels, bands are found at 210,000 and 203,000 daltons. This is in contrast to myosin from the wild-type, N2, which has a single heavy chain band at 210,000 daltons. Both heavy chains of E675 are found in body-wall muscle (Epstein, Waterston and Brenner, 1974). When native myosin from E675 is fractionated on hydroxyapatite, it is separated into myosin containing predominantly one or the other molecular weight heavy chain and myosin containing a mixture of the heavy chains. Comparison of the CNBr fragments of myosin that contains predominantly 210,000 dalton heavy chains with those of myosin that contains predominantly 203,000 dalton heavy chains reveals multiple differences. These differences are not explained by the difference in molecular weight of the heavy chains, but may be explained if each type of heavy chain is the product of a different structural gene. Furthermore, because there are fractions which exhibit >60% 210,000 or >60% 203,000 dalton heavy chain, there is myosin which is homogeneous for each of the heavy chains. Although N2 myosin has only a single molecular weight heavy chain, it too is fractionated by hydroxyapatite. By comparing the CNBr fragments of different myosin fractions, we show that N2, like E675, has two kinds of heavy chains. E190, a body-wall muscle-defective mutant in the same complementation group as E675, is lacking the myosin heavy chain affected by the e675 mutation. This property has allowed us to determine by co-purification of labeled El90 myosin in the presence of excess, unlabeled E675 myosin that most, if not all, of the myosin that contains two different molecular weight heavy chains is due to the formation of complexes between homogeneous myosins and not to a heterogeneous myosin. Introduction Starr and Offer (1973) reported that heavy chains of rabbit fast skeletal muscle myosin had two different
in Body-Wall
Muscle of
amino-terminal amino acid sequences. Although alternative hypotheses were considered, they proposed as the most probable explanation that there are at least two structural genes for rabbit fast skeletal muscle myosin heavy chains. Epstein et al. (1974) in their studies on myosin from the body-wall muscle-defective mutant E675 of the nematode, Caenorhabditis elegans, also found evidence for myosin heavy chain multiplicity in a single muscle type. SDS-polyacrylamide gel electrophoresis of actomyosin extracts from E675 revealed two different myosin heavy chains which were assigned masses of 210,000 and 203,000 daltons. In contrast, actomyosin extracts from the wild-type, N2, showed only a single 210,000 dalton heavy chain. Both molecular weight heavy chains of E675 were located in body-wall muscle. As the most straightforward explanation of these observations, they proposed that the heavy chains of bodywall muscle myosin in wild-type C. elegans were specified by two nonallellic structural genes, and that the mutation in E675 resulted in the conversion of one of the 210,000 dalton gene products to 203,000 daltons. In this paper, we confirm this proposal of Epstein et al. (1974). Further, we show that each of the different body-wall muscle myosin heavy chains associates with a like heavy chain in the native myosin molecule, which is dimeric with respect to the heavy chains. Chromatography of purified E675 myosin on hydroxyapatite resolves myosins homogeneous with respect to either the 210,000 or the 203,000 dalton heavy chains. Comparison of the CNBr peptides of these heavy chains suggests that they are the products of different structural genes. Using the CNBr fragments to detect the different heavy chains, we show that both are present in the wild-type and that its myosin fractionates similarly on hydroxyapatite. Analysis of another mutant, E190, in the same gene as E675 shows that it lacks the homogeneous myosin affected by the e675 mutation. This property allowed us to determine, by co-purification of El90 and E675 myosins, whether fractions from the E675 hydroxyapatite chromatography containing myosin with both heavy chains were due to a heterogeneous myosin species (one which has one of each type of heavy chain). The results indicate that most, if not all, of this mixed material is due to complexes between the two homogeneous myosin species. Results The E675 Myosin Heavy Chains Co-purify Figure 1 D shows the two predominant heavy chains of purified E675 myosin which can be distinguished by SDS-polyacrylamide gel electrophoresis. There
Cdl 722
is also a minor band at 206,000 daltons which Epstein et al. (1974) found in both E675 and N2, and which is located in pharyngeal muscle. Because of its molecular weight, the 206,000 dalton species was putatively identified as a myosin heavy chain. Figure l A-l D show the heavy chain composition of E675 myosin at various stages of its purification. The ratio of 203,000 to 210,000 dalton bands as determined by densitometry of 35S-labeled polypeptides remains constant, within experimental error, during the purification, varying from 1.91 in the homogenate to 2.12 in the Sepharose-purified myosin with a mean and standard deviation of 1.96 t 0.05. The 206,000 dalton band varies between 7% and 10% of the total of the three bands. The ratio of the two predominant heavy chain bands varies from preparation to preparation, but the 203,000 dalton heavy chains is always the major species. The 203,000 to 210,000 dalton ratio was 1.77 ? 0.17 in five homogenates analyzed by densitometry of Coomassie brilliant blue stain or of 35Sautoradiographs. Separation of Myosins Homogeneous for the 203,000 and 210,000 Dalton Heavy Chains When native E675 myosin is chromatographed on hydroxyapatite, species homogeneous for the 203,000 and 210,000 dalton heavy chains are resolved. Figure 2A shows the chromatogram when 600 pg of Sepharose-purified E675 myosin are eluted with a linear phosphate gradient from 0.005 M to 0.5 M with constant 0.6 M KCI, 0.001 M dithiothreitol, 0.0005 M phenylmethylsulfonylfluoride and 0.1% Triton X-100 at a constant pH of 6.5. The profile is complex with three apparent regions: a leading shoulder, fractions 70-89 (region I); a central peak, fractions 92-96 (region II); and a trailing shoulder, fractions 98-110 (region Ill). Since the different molecular weight heavy chains are clearly resolved by SDS-polyacrylamide gel electrophoresis, we are able to quantitate the separation of the two chains by densitometry. Inspection of Figure 3, an autoradiograph of the SDS-polyacrylamide gels on the ?S-myosin from the hydroxyapatite fractions, reveals that region I contains myosin which has predominantly 210,000 dalton heavy chain and region III myosin containing predominantly 203,000 dalton heavy chain. Fractions 75-80 contain myosin that has >80% 210,000 dalton heavy chains, and fractions 100-I 10 contain myosin with >80% 203,000 dalton heavy chains. This demonstrates that native E675 myosin, before electrophoresis under denaturing conditions, contains molecules that are homogeneous for either the 210,000 or 203,000 dalton heavy chains. Figure 26 shows the fractionation of the 210,000 and 203,000 dalton chains separately. The peak fractions are 87 and 103, respectively. Assuming that
A
B
C
D
210 kd
FRONT
Figure
1. Co-purification
of E675 Myosin
Heavy
Chains
Autoradiograph of samples from the purification of Yi-E675 myosin electrophoresed on 4.5% polyacrylamide-SDS gels to show the 210,000 and 203,000 dalton heavy chain polypeptides. (A) the homogenate; (8) the actomyosin extract; (C) the 100,000 supernatant 4; (D) purified myosin. Purification fractions refer to Harris and Epstein (1977).
the separation of chains reflects the separation of three myosin populations-a 210,000 dalton homogeneous, a 203,000 dalton homogeneous and a mixed myosin-we estimate that 24% of the material is homogeneous for the 210,000 dalton heavy chain, 52% is homogeneous for the 203,000 dalton heavy chain and 15% can be represented as myosin containing the two heavy chains. The remaining 8% is 206,000 dalton material and has a peak fraction 74. Its co-purification with myosin and co-migration with myosin on hydroxyapatite lend further support for its identification as a pharyngeal myosin heavy chain. To confirm the identification of two homogeneous myosins, fractions from regions I and III were
Two
Nematode
Body-Wall
Myosins
723
I
14-
I
I
1
1
1
"d'
’
""1
A
12-
8 6 4
T6 -
I
2 0 1011
I ’ -
B
1 ’ ?
’
1 ’ 1
‘D
8642-
ing buffer for the chromatography. Upon rechromatography, this myosin elutes with a maximum at fraction 87 (Figure 2C). When myosin from region III (fractions 101-108) composed of 67% 203,000 dalton heavy chain is similarly treated, it rechromatographs with a maximum at fraction 103 (Figure 2D). These maxima correspond to the maxima of 210,000 and 203,000 dalton myosin heavy chains from the densitometric analysis of the original chromatography (Figure 2B). Thus the homogeneous myosins of E675 rechromatograph true. When myosin containing both molecular weight heavy chains from region II (fractions 92-97) was rechromatographed, it was clear that the 203,000 dalton homogeneous myosin was a major component. The second maximum of the rechromatography at fractions 98-103 corresponds to the elution position of the 203,000 dalton homogeneous myosin. Densitometric analysis of SDS-polyacrylamide gels of the rechromatographed fraction is consistent with this interpretation. However, the absence of 210,000 dalton homogeneous myosin in the rechromatography suggests that the heavy chains do not exchange between myosin molecules during hydroxyapatite chromatography.
ol 14 70
90 FB A:,b?d
10 -
7 ,o x
B 8
8642-
Figure
2. Hydroxyapatite
Chromatography
FRACTION NO. of E675 Myosin
Chromatograms of hydroxyapatite chromatography of %-E675 myosin and rechromatography of fractions of the initial chromatography. Hydroxyapatite columns were run at 2-3 ml/hr. and 1 ml samples were collected. (A) chromatography of E675 myosin: 50 AI samples from each fraction counted as in Experimental Procedures. (6) densitometric analysis of the chromatography based on gels of each of the fractions (Figure 3). (O-O) represents the 210,000 dalton heavy chain; (O-O) the 203,000 dalton heavy chain. Ratios of the chains were determined by densitometry. and the fraction of counts due to each was plotted. (C). (D) and (E) are the rechromatography of fractions 73-81. 92-97 and 101-106, respectively. 100 ~1 samples from each fraction were counted as in Experimental Procedures.
rechromatographed. Myosin from region I (fractions 73-81) composed of 84% 210,000 dalton myosin heavy chain was dialyzed against the start-
Both Homogeneous Myosins Contain 18,000 and 16,000 Dalton Light Chains Harris and Epstein (1977) showed that two light chains of 18,000 and 16,000 daltons are observed in SDS-polyacrylamide gel electrophoresis of N2 myosin. Both these light chains are present in E675 myosin. Figure 4 shows the light chains of Sepharose-purified myosin and those of myosin from the fractions of regions I and Ill which were pooled for rechromatography. The material in region I contains some diffuse material of mass
