- Email: [email protected]
Extraction of proteins from Saccharomyces cerevisiae ribosomes under nondenaturing conditions
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 237, No. 2, March, pp. 292-299, 1985
Extraction of Proteins from Saccharomyces cerevisiae Ribosomes under Nondenaturing Conditions JOHN C. LEE,’ ROCHELLE ANDERSON, YEE CHUN YEH, AND PAUL HOROWITZ Department
of Biochemistry,
The University
Received August
of Texas Health, Science Center, San Antonio,
Texas 78284
15. 1984, and in revised form October 23, 1984
The differential sensitivity of ribosomal proteins to removal by salts has been studied. Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH&l (0.74-3.56 M) for a defined time (20 min) at 0°C. The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly. Proteins extracted under each salt condition were analyzed for composition by twodimensional polyacrylamide gel electrophoresis. The relative quantity of each protein was determined. Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction. Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle. The kinetics of protein release from both subunits at a single NH&l concentration (0.74 M) were also studied. Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins. However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts. Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment. Q 1985 Academic PRSS. h.
cles can be regenerated from proteindeficient core particles and proteins (410). Cox has recently reported a procedure for generating ribosomal core particles from the small ribosomal subunits of rabbit reticulocytes under nondenaturing conditions (8). These core particles can subsequently be used to regenerate biologically active ribosomes. In the present investigation suitable conditions for successive removal of yeast ribosomal components with increasing concentrations of NH&l under nondenaturing conditions were defined. Proteins extracted under each salt condition were identified. Most proteins could be removed from the ribosomal particles and were enriched in a single fraction.
Ribosomes are complex structures consisting of numerous RNA and protein species. One experimental approach in establishing the functional role of individual components is successive splittingoff of individual components from ribosomal particles by washing these particles with increasing concentrations of salts and subsequently reconstituting the ribosomal particles from these fractions. Invaluable information has been obtained on the bacterial ribosomes using this approach [for review (l-3)]. Eucaryotic ribosomes do not respond in the same way as procaryotic ribosomes. In only a few cases, biologically active ribosomal parti’ To whom correspondence
should be addressed.
0003-9861185 $3.00 Copyright 0 1995 by Academic Press. Inc. All rights of reproduction in any form reserved.
292
CONTROLLED EXPERIMENTAL
DISASSEMBLY
PROCEDURES
Yeast culture and ribosme preparation Sacchar@ myces cerevisiae were grown and collected in late log phase. Ribosomal subunits were isolated by zonal centrifugation in glycerol gradients as previously described by Lee et al. (11). Purity of the ribosomal subunits was checked by analytical centrifugation in glycerol gradients and by RNA analysis. Only those ribosomal preparations of >96% purity were used for the studies reported here. Eztractim procedures. The basic extraction procedure was based on the results of Cox (8), which showed that the ribosomal RNAs provided with a minimum molar ratio of Mgz+ to NH: at 1:40 will maintain their “native” conformation. The present protocol involved serial extraction of ribosomes by gentle shaking in a series of buffers containing increasing concentrations of NH&l but maintaining the molar ratio of Mgz’ and NH: at 1:40. The subparticles generated were separated from the extracted proteins by centrifugation and used as starting material for the next extraction. Purified yeast 60 S or 40 S subunits (in Buffer I: 50 mM Tris, pH 7.7, 1 mM DTT,2 0.1 mM EDTA, 600 mM KCl, 8 mM MgCl, at a concentration of 100 A 260“,,, units/ml) were used for each extraction series. An aliquot was removed before the salt extraction and the total protein complement was isolated and analyzed by two-dimensional polyacrylamide gel electrophoresis. The remaining ribosomal sample was mixed slowly with Buffer II (4.45 M NH&I, 100 mM MgClz, 20 XIIM Tris, pH 7.5, 10 mM mercaptoethanol) to give the desired final concentration of NH&l. The mixture was shaken gently at 0°C for 20 min followed by centrifugation in a Beckman 40 rotor at 38,000 rpm for 2.5 h at 4°C. The supernatant was removed immediately to minimize resolubilization of the pellet. An aliquot of the supernatant was removed for RNA analysis. The remaining solution was dialyzed against acetic acid (6%) containing 20 mM mercaptoethanol for 24 h at 4°C with two buffer changes. The pellet was suspended in equal volume of Buffer I and mixed with a sufficient volume of Buffer II to give the desired final concentration of NH&l. After centrifugation, the supernatant obtained was divided into two equal aliquots-one was dialyzed against acetic acid for subsequent protein analysis and the other was extracted with phenol for subsequent RNA analysis. The pellet obtained after extraction with 3.56~ NH&I was suspended in Buffer I and extracted with acetic acid as described by Hardy et al. (12). The protein extract was also dialyzed against acetic acid. The proteins present in each extraction were lyophilized to dryness. Protein analysis. Proteins present in each extraction were dissolved in the sample solution of the
’ Abbreviation
used: DTT, dithiothreitol.
OF YEAST
293
RIBOSOME
two-dimensional electrophoresis system previously described (13). An aliquot was removed for protein determination (14). The remaining protein samples were analyzed by two-dimensional gel electrophoresis (13). Proteins were stained with Coomassie blue and numbered as described (15). In order to determine the proportion of each protein that was present in each extraction condition, each protein spot on the gel was excised and eluted with 0.5 ml 25% pyridine (16). The optical density of the solution at 635 nm was determined. To aid in the comparison of data from different experiments and to compensate for the varying intensity of Coomassie blue stain for different proteins, the intensity of Coomassie blue stain of the extracted proteins was normalized against that for the same protein in a control sample. Isolation and gel analysis of RNA. RNA was extracted with phenol and precipitated with ethanol as described previously (11). The RNA pellet was dissolved in 6M urea and the amount of RNA present in each sample was determined spectrophotometrically at 260 nm. To determine the species of RNA present, RNA samples were analyzed by electrophoresis on polyacrylamide gels (11). RESULTS
Figure 1 shows the amount of protein extracted from 60 S or 40 S ribosomal
80ap
1.0
2.0
3.0
4.0
Nl$CI Concentration. M
FIG. 1. Cumulative amounts of RNA or protein released from yeast ribosomal subunits by serial washing of the subunits with buffers containing increasing concentrations of NH&l under nondenaturing conditions. Proteins from 60 S (0) and 40 S (X); RNA from 60 S (0) and 40 S (A).
LEE
ET AL.
-,
._ -_
i,. Y
FIG. 2. Analysis of 60 S and 40 S ribosomal subunit proteins extracted by increasing concentrations of NH&I. Extracted proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. First dimension, left to right (+ - -); second dimension, top to bottom (- +). A-F represent electrophoretic profiles of proteins from 40 S subunits extracted with 0, 0.74, 1.48,2.96, and 3.56 M NH&l, and the final residual core fraction. G-L represent the electrophoretic profiles of proteins from the 60 S subunits extracted with 0, 0.74, 1.48, 2.96, and 3.56 M NH&l, and the final residual core fraction.
subunits upon treatment with different concentrations of NH&l. The shape of the curves of protein released for both subunits appears to be identical. Also shown in Fig. 1 is the amount of RNA released from the subunits under these extraction conditions. Greater than 95% of the rRNAs remained in the final residual pellet (after 3.56 M NH&l extraction).
The protein composition of each fraction and of the final residual core fraction was analyzed by two-dimensional electrophoresis on polyacrylamide gels (Fig. 2). Based on the intensity of the Coomassie blue stain, most proteins appeared not to be completely removed from the ribosomal particle under any one condition, but appeared to partition differentially among
CONTROLLED
DISASSEMBLY
OF YEAST
RIBOSOME
295
FIG. 2-Continued.
several conditions. In order to analyze more closely the distribution of individual proteins, the Coomassie blue stain was normalized as described under Experimental Procedures. The patterns of distribution of several representative proteins from both ribosomal subunits are shown in Fig. 3. This type of analysis revealed that most proteins were preferentially extracted into one fraction in which they were enriched. Several proteins (e.g., S3, L6, L12, and L16) were not enriched in any particular fraction. Although most proteins could be solubilized, several
proteins (e.g., Sl and L3) remained predominantly or exclusively with the core fraction even after treatment with 3.56 M NH&l. The enriched proteins of each fraction are summarized in Table I. The kinetics of release of ribosomal proteins from the 40 S ribosomal subunits at a single NH&l concentration (0.74 M) were also studied. The release of proteins was time dependent (Fig. 4). More of the same proteins (such as 54, S14, and 229) which were classified as enriched proteins in this salt fraction (Table I) were released. However, prolonged treatment (240
296
LEE
0
1.0 NH4Cl
2.0
3.0
Concentration.
4.0
ET AL.
L26) which were classified as enriched proteins in this salt fraction (Table I) were released, but also additional proteins (such as L22) began to be extracted even at 60 min. After 240 min of extraction, additional proteins (such as L21, L23, L29, and L43) which were classified as proteins enriched in the 2.96 and 3.56 M NH&l wash fractions (Table I) began to be extracted (Fig. 5). By 240 min 33 of the 45 proteins from the 60 S ribosomal subunits were detectable in varying amounts. To assess the type of ribosomal RNA released from the ribosomal subunits under each extraction condition, each fraction was analyzed by electrophoresis on polyacrylamide gels. Figure 6 shows that 5 S RNA began to be released from the 60 S subunits upon exposure of the subunits to 0.74 M NH&l. The release continued as the ribosomal subunits were exposed to 1.48 and 2.96 M NH&l. Only very small amounts of the 5 S RNA remained in the ribosomal subparticles after the 2.96 M NH&l extraction. On the other hand, the other ribosomal RNA species (5.8 S, 18 S, and 26 S) remained exclusively in the final residual core particles even after 3.56 M NHICl extraction.
residual core M
FIG. 3. Distribution of proteins from yeast 60 S and 40 S ribosomal subunits among the various NH&I washing fractions and the final residual core fraction.
min) of subunits generated a few additional proteins (such as S6 and S28) which were classified as proteins enriched in fractions extracted with higher concentrations of NH&l. In fact, 15 out of the 32 proteins from the 40 S ribosomal subunits were detectable, in varying amounts, in the 240-min extraction. In contrast to the 40 S subunits, proteins from the 60 S subunits were relatively more susceptible to salt extraction (Fig. 5). Not only more of the same proteins (such as L7, L15, and
DISCUSSION
The present study demonstrates the feasibility of specific extraction of proteins from yeast ribosomal subunits by sequen-
TABLE PROTEINS
Fraction
ENRICHED
IN NH&I
WASH
I
FRACTIONS
60 S subunit
AND THE RESIDUAL
proteins
CORE FRACTIONS
40 S subunit
proteins
0.74 M NH&l
wash
Lla, L7, LW9, L15, L17, L26, L30, L34, L37, L38, L47
S4, Sll, S14, S19, S20, S24, S26a, S31, S37
1.48 M NH&I
wash
LlO, L36, L41
S6, S28
2.96 M NH&l
wash
L18, L22, L33, L39, L42, L43, L46
SlO, S26, S32
3.56 M NH&l
wash
L2, L4, L5, L20, L21, L23, L29, L31, L32, L35
SO, S12, S16, S27, 533
L3, L13, L17a, L19, L25
Sl, S2, S13, S18, S27a
Residual
Core
Note. Proteins not enriched in any particular fractions: L6, L12, L16, and S3. These classifications based on four independent determinations on four separate preparations of ribosomal subunits.
are
CONTROLLED
DISASSEMBLY
OF YEAST
RIBOSOME
297
viously shown to maintain the rRNAs in their “native” conformation (8,17). Under these conditions, most proteins from the 60 S and the 40 S ribosomal subunits of S. cerevisiae were preferentially extracted into one or two NH&l fractions. Although our data cannot distinguish between the removal of individual proteins and of small aggregates from the ribosome, the findings do show that there is a differential sensitivity of ribosomal proteins to removal by salts and demonstrate differential stability of protein interactions in the assembled ribosomal particles.
FIG. 4. Analysis of 40 S ribosomal subunit proteins extracted by 0.74 M NH&I as a function of time. Extracted proteins were analyzed as described in Fig. 2. (A) 30; (B) 60; (C) 240 min of extraction.
tially washing the ribonucleoprotein particles with a series of solutions containing increasing concentrations of NH&l. The magnesium concentration was increased proportionally to maintain a molar ratio of Mg2+ to NH: at 1:40, a condition pre-
FIG. 5. Analysis of 60 S ribosomal subunit proteins extracted by 0.74 M NH&l as a function of time. Extracted proteins were analyzed as described in Fig. 2 (A) 60; (B) 120; (C) 240 min of extraction.
298
LEE
ET AL.
Based on in viva labeling kinetics of individual proteins, Kruiswijk et aL (20) classified many of the yeast ribosomal proteins into two broad groups (early or late) according to their time of association with the ribonucleoprotein particles during ribosomal maturation. When compared with our data, proteins in the final 1 2 3 4 5 6 residual core fraction derived from either 60 S or the 40 S ribosomal subunits are FIG. 6. Electrophoretic profiles of RNA present in classified as early assembled proteins (Tathe various NH&l extraction fractions and in the ble I). These data raise the possibility final residual core fraction. Lanes 1-6 represent RNA in the 0.74, 1.48, 2.96, and 3.56 M NH&l extracts, that the order of extraction may reflect the control fraction, and the residual core fraction. the sequence of protein assembly in vivo during ribosome maturation. In addition, it is noteworthy that protein L8/9, a readily extracted protein, is among those No detectable small RNA fragments were observed in the NH&l fractions and proteins which are “exchangeable” in vivo, the core fraction. The 5 S rRNA species i.e., they are found to be associated with as well as its binding protein (Lla) were ribosomes in the absence of new protein released from the ribosomal particles un- synthesis (21). In summary, the present investigation der relatively low salt conditions. This demonstrates the potential usefulness of finding may imply that the interactions between the 5 S RNA (or its binding pro- serial NH&l extraction procedures for tein) and the remaining ribosomal particle the controlled disassembly of yeast ribosomes under nondenaturing conditions. or the interactions between the 5 S RNAThis protocol will generate a series of protein Lla complex and the remaining particle are predominantly ionic in nature. fractions, each specifically enriched for It has been previously reported by Nazar several proteins. Each fraction can be et al. (18) that treatment of yeast 60 S used as a source of ribosomal proteins for ribosomes with 25 mM EDTA released a immunological, functional, and structural ribonucleoprotein complex which consists studies. This procedure can potentially generate information about the organiof the 5 S RNA species and its binding protein. Our data cannot rule out the zation of protein and RNA in ribosomal possibility that these two components are structure such as identification of candidates for RNA-binding proteins as well released as a complex. On the contrary, as their general order of assembly during the 5.8 S RNA remained associated with the 3.56 M or the residual core fraction as ribosomal maturation. In addition, it is well as all of the previously reported 5.8 hoped that the present results will be S RNA-binding proteins (L12, L19, L20, useful in future attempts to reassemble L23, L25, L29, L30, L31, L33, L35, and L39) active eucaryotic ribosomes. with the exception of protein L30. Protein REFERENCES L30 is among those that will not bind to the 5.8 S RNA as an individual protein 1. NOMURA, M. (1973) Science (Washingta, D. C) even though it is a member of the ribo179, 864-873. nucleoprotein complex (11). Several pro2. NOMURA, M., AND HELD, W. A. (1974) in Ribosomes (Nomura, M., Tissieres, A., and Lengyel, teins (e.g., SlO, S13, S27, L2/3, L5, L19/ P., eds.), pp. 193-223, Cold Spring Harbor 20, L22, L23, L42, and L43) which were Laboratory, Cold Spring Harbor, New York. detected in the high-salt wash fractions 3. NIERHAUS, K. H. (1979) in Ribosomes: Structure, (2.96 and 3.56 M) and the final residual Function, and Genetics (Chambliss, G., Craven, core fraction were classified by Reyes et G. R., Davis, J., Davis, K., Kahan, L., and al. (19) to be RNA-binding proteins in the Nomura, M., eds.), pp. 267-294, University ribosomes. Park Press, Baltimore.
CONTROLLED
DISASSEMBLY
4. REBOLJD, A. M., HAMILTON, M. G., AND PETERMAN, M. L. (1969) Biochemistry 8, 843-850. 5. GRUMMT, F., AND BIELKA, H. (1970) Biochim
Biophys. Acta 199, 540-542. 6. TERAO,
K., AND OGATA,
K. (1971)
B&him.
Biw
phys. Acta 254,278-295. 7. REYES,
R., VAZQUEZ,
D., AND BALLESTA,
J. P. G.
(1976) Biochim. Biophys. Acta 433, 317-332. 8. Cox, R. A. (1981) B&hem. .I 194,931-939. 9. VIOQUE,
A., PINTOR-TORO,
A., AND PALACIAN,
E.
(1982) J. Biol Chem. 257, 6477-6480. 10. BIELKA, H., AND STAHL, J. (1978) Biochem
Ser.
One 18, 111-168. 11. LEE, J. C., HENRY, B., AND YEH, Y. C. (1983) Biol. Chem 258, 854-858. 12. HARDY, AND
J.
S. J. S., KURLAND, C. G., VOYNOW, P., MORA, G. (1969) Biochemistry 8, 2897-
2905. 13. OTAKA,
E.,
AND
KOBATA,
Genet. 162,259-268.
K.
(1978)
Mel
Gen.
OF YEAST
299
RIBOSOME
14. LOWRY, 0. H., ROSENBROUGH, N., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem 193,
265-275. 15. MICHEL, S., TRAUT, R. R., AND LEE, J. C. (1983) Mol. Gen. Genet. 191,251-256. 16. FENNER, C., TRAUT, R. R., MASON, D. T., AND WIKMAN-COFFELT, J. (1975) Anal B&hem. 63, 595-602. 17. Cox, R. A., AND HIRST, W. (1976) B&hem. J. 160, 505-519. 18. NAZAR,
R., YAGUCHI,
M., WILLICK,
C. F., AND ROY, C. (1979) Eur. 102,573-582.
G. E., ROLLIN,
J. B&hem.
19. REYES, R., VAZQUEZ, D., AND BALLESTA, J. P. G. (1978) B&him. Biophys. Acta 521, 229-234. 20. KRIJISWIJK, T., PLANTA, R. J., AND KROP, J. M. (1978) Biochim. Biophvs. Acta 517, 378-389. 21. ZINKER,
S., AND WARNER,
Chem. 251, 1799-1805.
J. R. (1976)
J. Biol
Extraction of Proteins from Saccharomyces cerevisiae Ribosomes under Nondenaturing Conditions JOHN C. LEE,’ ROCHELLE ANDERSON, YEE CHUN YEH, AND PAUL HOROWITZ Department
of Biochemistry,
The University
Received August
of Texas Health, Science Center, San Antonio,
Texas 78284
15. 1984, and in revised form October 23, 1984
The differential sensitivity of ribosomal proteins to removal by salts has been studied. Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH&l (0.74-3.56 M) for a defined time (20 min) at 0°C. The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly. Proteins extracted under each salt condition were analyzed for composition by twodimensional polyacrylamide gel electrophoresis. The relative quantity of each protein was determined. Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction. Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle. The kinetics of protein release from both subunits at a single NH&l concentration (0.74 M) were also studied. Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins. However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts. Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment. Q 1985 Academic PRSS. h.
cles can be regenerated from proteindeficient core particles and proteins (410). Cox has recently reported a procedure for generating ribosomal core particles from the small ribosomal subunits of rabbit reticulocytes under nondenaturing conditions (8). These core particles can subsequently be used to regenerate biologically active ribosomes. In the present investigation suitable conditions for successive removal of yeast ribosomal components with increasing concentrations of NH&l under nondenaturing conditions were defined. Proteins extracted under each salt condition were identified. Most proteins could be removed from the ribosomal particles and were enriched in a single fraction.
Ribosomes are complex structures consisting of numerous RNA and protein species. One experimental approach in establishing the functional role of individual components is successive splittingoff of individual components from ribosomal particles by washing these particles with increasing concentrations of salts and subsequently reconstituting the ribosomal particles from these fractions. Invaluable information has been obtained on the bacterial ribosomes using this approach [for review (l-3)]. Eucaryotic ribosomes do not respond in the same way as procaryotic ribosomes. In only a few cases, biologically active ribosomal parti’ To whom correspondence
should be addressed.
0003-9861185 $3.00 Copyright 0 1995 by Academic Press. Inc. All rights of reproduction in any form reserved.
292
CONTROLLED EXPERIMENTAL
DISASSEMBLY
PROCEDURES
Yeast culture and ribosme preparation Sacchar@ myces cerevisiae were grown and collected in late log phase. Ribosomal subunits were isolated by zonal centrifugation in glycerol gradients as previously described by Lee et al. (11). Purity of the ribosomal subunits was checked by analytical centrifugation in glycerol gradients and by RNA analysis. Only those ribosomal preparations of >96% purity were used for the studies reported here. Eztractim procedures. The basic extraction procedure was based on the results of Cox (8), which showed that the ribosomal RNAs provided with a minimum molar ratio of Mgz+ to NH: at 1:40 will maintain their “native” conformation. The present protocol involved serial extraction of ribosomes by gentle shaking in a series of buffers containing increasing concentrations of NH&l but maintaining the molar ratio of Mgz’ and NH: at 1:40. The subparticles generated were separated from the extracted proteins by centrifugation and used as starting material for the next extraction. Purified yeast 60 S or 40 S subunits (in Buffer I: 50 mM Tris, pH 7.7, 1 mM DTT,2 0.1 mM EDTA, 600 mM KCl, 8 mM MgCl, at a concentration of 100 A 260“,,, units/ml) were used for each extraction series. An aliquot was removed before the salt extraction and the total protein complement was isolated and analyzed by two-dimensional polyacrylamide gel electrophoresis. The remaining ribosomal sample was mixed slowly with Buffer II (4.45 M NH&I, 100 mM MgClz, 20 XIIM Tris, pH 7.5, 10 mM mercaptoethanol) to give the desired final concentration of NH&l. The mixture was shaken gently at 0°C for 20 min followed by centrifugation in a Beckman 40 rotor at 38,000 rpm for 2.5 h at 4°C. The supernatant was removed immediately to minimize resolubilization of the pellet. An aliquot of the supernatant was removed for RNA analysis. The remaining solution was dialyzed against acetic acid (6%) containing 20 mM mercaptoethanol for 24 h at 4°C with two buffer changes. The pellet was suspended in equal volume of Buffer I and mixed with a sufficient volume of Buffer II to give the desired final concentration of NH&l. After centrifugation, the supernatant obtained was divided into two equal aliquots-one was dialyzed against acetic acid for subsequent protein analysis and the other was extracted with phenol for subsequent RNA analysis. The pellet obtained after extraction with 3.56~ NH&I was suspended in Buffer I and extracted with acetic acid as described by Hardy et al. (12). The protein extract was also dialyzed against acetic acid. The proteins present in each extraction were lyophilized to dryness. Protein analysis. Proteins present in each extraction were dissolved in the sample solution of the
’ Abbreviation
used: DTT, dithiothreitol.
OF YEAST
293
RIBOSOME
two-dimensional electrophoresis system previously described (13). An aliquot was removed for protein determination (14). The remaining protein samples were analyzed by two-dimensional gel electrophoresis (13). Proteins were stained with Coomassie blue and numbered as described (15). In order to determine the proportion of each protein that was present in each extraction condition, each protein spot on the gel was excised and eluted with 0.5 ml 25% pyridine (16). The optical density of the solution at 635 nm was determined. To aid in the comparison of data from different experiments and to compensate for the varying intensity of Coomassie blue stain for different proteins, the intensity of Coomassie blue stain of the extracted proteins was normalized against that for the same protein in a control sample. Isolation and gel analysis of RNA. RNA was extracted with phenol and precipitated with ethanol as described previously (11). The RNA pellet was dissolved in 6M urea and the amount of RNA present in each sample was determined spectrophotometrically at 260 nm. To determine the species of RNA present, RNA samples were analyzed by electrophoresis on polyacrylamide gels (11). RESULTS
Figure 1 shows the amount of protein extracted from 60 S or 40 S ribosomal
80ap
1.0
2.0
3.0
4.0
Nl$CI Concentration. M
FIG. 1. Cumulative amounts of RNA or protein released from yeast ribosomal subunits by serial washing of the subunits with buffers containing increasing concentrations of NH&l under nondenaturing conditions. Proteins from 60 S (0) and 40 S (X); RNA from 60 S (0) and 40 S (A).
LEE
ET AL.
-,
._ -_
i,. Y
FIG. 2. Analysis of 60 S and 40 S ribosomal subunit proteins extracted by increasing concentrations of NH&I. Extracted proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. First dimension, left to right (+ - -); second dimension, top to bottom (- +). A-F represent electrophoretic profiles of proteins from 40 S subunits extracted with 0, 0.74, 1.48,2.96, and 3.56 M NH&l, and the final residual core fraction. G-L represent the electrophoretic profiles of proteins from the 60 S subunits extracted with 0, 0.74, 1.48, 2.96, and 3.56 M NH&l, and the final residual core fraction.
subunits upon treatment with different concentrations of NH&l. The shape of the curves of protein released for both subunits appears to be identical. Also shown in Fig. 1 is the amount of RNA released from the subunits under these extraction conditions. Greater than 95% of the rRNAs remained in the final residual pellet (after 3.56 M NH&l extraction).
The protein composition of each fraction and of the final residual core fraction was analyzed by two-dimensional electrophoresis on polyacrylamide gels (Fig. 2). Based on the intensity of the Coomassie blue stain, most proteins appeared not to be completely removed from the ribosomal particle under any one condition, but appeared to partition differentially among
CONTROLLED
DISASSEMBLY
OF YEAST
RIBOSOME
295
FIG. 2-Continued.
several conditions. In order to analyze more closely the distribution of individual proteins, the Coomassie blue stain was normalized as described under Experimental Procedures. The patterns of distribution of several representative proteins from both ribosomal subunits are shown in Fig. 3. This type of analysis revealed that most proteins were preferentially extracted into one fraction in which they were enriched. Several proteins (e.g., S3, L6, L12, and L16) were not enriched in any particular fraction. Although most proteins could be solubilized, several
proteins (e.g., Sl and L3) remained predominantly or exclusively with the core fraction even after treatment with 3.56 M NH&l. The enriched proteins of each fraction are summarized in Table I. The kinetics of release of ribosomal proteins from the 40 S ribosomal subunits at a single NH&l concentration (0.74 M) were also studied. The release of proteins was time dependent (Fig. 4). More of the same proteins (such as 54, S14, and 229) which were classified as enriched proteins in this salt fraction (Table I) were released. However, prolonged treatment (240
296
LEE
0
1.0 NH4Cl
2.0
3.0
Concentration.
4.0
ET AL.
L26) which were classified as enriched proteins in this salt fraction (Table I) were released, but also additional proteins (such as L22) began to be extracted even at 60 min. After 240 min of extraction, additional proteins (such as L21, L23, L29, and L43) which were classified as proteins enriched in the 2.96 and 3.56 M NH&l wash fractions (Table I) began to be extracted (Fig. 5). By 240 min 33 of the 45 proteins from the 60 S ribosomal subunits were detectable in varying amounts. To assess the type of ribosomal RNA released from the ribosomal subunits under each extraction condition, each fraction was analyzed by electrophoresis on polyacrylamide gels. Figure 6 shows that 5 S RNA began to be released from the 60 S subunits upon exposure of the subunits to 0.74 M NH&l. The release continued as the ribosomal subunits were exposed to 1.48 and 2.96 M NH&l. Only very small amounts of the 5 S RNA remained in the ribosomal subparticles after the 2.96 M NH&l extraction. On the other hand, the other ribosomal RNA species (5.8 S, 18 S, and 26 S) remained exclusively in the final residual core particles even after 3.56 M NHICl extraction.
residual core M
FIG. 3. Distribution of proteins from yeast 60 S and 40 S ribosomal subunits among the various NH&I washing fractions and the final residual core fraction.
min) of subunits generated a few additional proteins (such as S6 and S28) which were classified as proteins enriched in fractions extracted with higher concentrations of NH&l. In fact, 15 out of the 32 proteins from the 40 S ribosomal subunits were detectable, in varying amounts, in the 240-min extraction. In contrast to the 40 S subunits, proteins from the 60 S subunits were relatively more susceptible to salt extraction (Fig. 5). Not only more of the same proteins (such as L7, L15, and
DISCUSSION
The present study demonstrates the feasibility of specific extraction of proteins from yeast ribosomal subunits by sequen-
TABLE PROTEINS
Fraction
ENRICHED
IN NH&I
WASH
I
FRACTIONS
60 S subunit
AND THE RESIDUAL
proteins
CORE FRACTIONS
40 S subunit
proteins
0.74 M NH&l
wash
Lla, L7, LW9, L15, L17, L26, L30, L34, L37, L38, L47
S4, Sll, S14, S19, S20, S24, S26a, S31, S37
1.48 M NH&I
wash
LlO, L36, L41
S6, S28
2.96 M NH&l
wash
L18, L22, L33, L39, L42, L43, L46
SlO, S26, S32
3.56 M NH&l
wash
L2, L4, L5, L20, L21, L23, L29, L31, L32, L35
SO, S12, S16, S27, 533
L3, L13, L17a, L19, L25
Sl, S2, S13, S18, S27a
Residual
Core
Note. Proteins not enriched in any particular fractions: L6, L12, L16, and S3. These classifications based on four independent determinations on four separate preparations of ribosomal subunits.
are
CONTROLLED
DISASSEMBLY
OF YEAST
RIBOSOME
297
viously shown to maintain the rRNAs in their “native” conformation (8,17). Under these conditions, most proteins from the 60 S and the 40 S ribosomal subunits of S. cerevisiae were preferentially extracted into one or two NH&l fractions. Although our data cannot distinguish between the removal of individual proteins and of small aggregates from the ribosome, the findings do show that there is a differential sensitivity of ribosomal proteins to removal by salts and demonstrate differential stability of protein interactions in the assembled ribosomal particles.
FIG. 4. Analysis of 40 S ribosomal subunit proteins extracted by 0.74 M NH&I as a function of time. Extracted proteins were analyzed as described in Fig. 2. (A) 30; (B) 60; (C) 240 min of extraction.
tially washing the ribonucleoprotein particles with a series of solutions containing increasing concentrations of NH&l. The magnesium concentration was increased proportionally to maintain a molar ratio of Mg2+ to NH: at 1:40, a condition pre-
FIG. 5. Analysis of 60 S ribosomal subunit proteins extracted by 0.74 M NH&l as a function of time. Extracted proteins were analyzed as described in Fig. 2 (A) 60; (B) 120; (C) 240 min of extraction.
298
LEE
ET AL.
Based on in viva labeling kinetics of individual proteins, Kruiswijk et aL (20) classified many of the yeast ribosomal proteins into two broad groups (early or late) according to their time of association with the ribonucleoprotein particles during ribosomal maturation. When compared with our data, proteins in the final 1 2 3 4 5 6 residual core fraction derived from either 60 S or the 40 S ribosomal subunits are FIG. 6. Electrophoretic profiles of RNA present in classified as early assembled proteins (Tathe various NH&l extraction fractions and in the ble I). These data raise the possibility final residual core fraction. Lanes 1-6 represent RNA in the 0.74, 1.48, 2.96, and 3.56 M NH&l extracts, that the order of extraction may reflect the control fraction, and the residual core fraction. the sequence of protein assembly in vivo during ribosome maturation. In addition, it is noteworthy that protein L8/9, a readily extracted protein, is among those No detectable small RNA fragments were observed in the NH&l fractions and proteins which are “exchangeable” in vivo, the core fraction. The 5 S rRNA species i.e., they are found to be associated with as well as its binding protein (Lla) were ribosomes in the absence of new protein released from the ribosomal particles un- synthesis (21). In summary, the present investigation der relatively low salt conditions. This demonstrates the potential usefulness of finding may imply that the interactions between the 5 S RNA (or its binding pro- serial NH&l extraction procedures for tein) and the remaining ribosomal particle the controlled disassembly of yeast ribosomes under nondenaturing conditions. or the interactions between the 5 S RNAThis protocol will generate a series of protein Lla complex and the remaining particle are predominantly ionic in nature. fractions, each specifically enriched for It has been previously reported by Nazar several proteins. Each fraction can be et al. (18) that treatment of yeast 60 S used as a source of ribosomal proteins for ribosomes with 25 mM EDTA released a immunological, functional, and structural ribonucleoprotein complex which consists studies. This procedure can potentially generate information about the organiof the 5 S RNA species and its binding protein. Our data cannot rule out the zation of protein and RNA in ribosomal possibility that these two components are structure such as identification of candidates for RNA-binding proteins as well released as a complex. On the contrary, as their general order of assembly during the 5.8 S RNA remained associated with the 3.56 M or the residual core fraction as ribosomal maturation. In addition, it is well as all of the previously reported 5.8 hoped that the present results will be S RNA-binding proteins (L12, L19, L20, useful in future attempts to reassemble L23, L25, L29, L30, L31, L33, L35, and L39) active eucaryotic ribosomes. with the exception of protein L30. Protein REFERENCES L30 is among those that will not bind to the 5.8 S RNA as an individual protein 1. NOMURA, M. (1973) Science (Washingta, D. C) even though it is a member of the ribo179, 864-873. nucleoprotein complex (11). Several pro2. NOMURA, M., AND HELD, W. A. (1974) in Ribosomes (Nomura, M., Tissieres, A., and Lengyel, teins (e.g., SlO, S13, S27, L2/3, L5, L19/ P., eds.), pp. 193-223, Cold Spring Harbor 20, L22, L23, L42, and L43) which were Laboratory, Cold Spring Harbor, New York. detected in the high-salt wash fractions 3. NIERHAUS, K. H. (1979) in Ribosomes: Structure, (2.96 and 3.56 M) and the final residual Function, and Genetics (Chambliss, G., Craven, core fraction were classified by Reyes et G. R., Davis, J., Davis, K., Kahan, L., and al. (19) to be RNA-binding proteins in the Nomura, M., eds.), pp. 267-294, University ribosomes. Park Press, Baltimore.
CONTROLLED
DISASSEMBLY
4. REBOLJD, A. M., HAMILTON, M. G., AND PETERMAN, M. L. (1969) Biochemistry 8, 843-850. 5. GRUMMT, F., AND BIELKA, H. (1970) Biochim
Biophys. Acta 199, 540-542. 6. TERAO,
K., AND OGATA,
K. (1971)
B&him.
Biw
phys. Acta 254,278-295. 7. REYES,
R., VAZQUEZ,
D., AND BALLESTA,
J. P. G.
(1976) Biochim. Biophys. Acta 433, 317-332. 8. Cox, R. A. (1981) B&hem. .I 194,931-939. 9. VIOQUE,
A., PINTOR-TORO,
A., AND PALACIAN,
E.
(1982) J. Biol Chem. 257, 6477-6480. 10. BIELKA, H., AND STAHL, J. (1978) Biochem
Ser.
One 18, 111-168. 11. LEE, J. C., HENRY, B., AND YEH, Y. C. (1983) Biol. Chem 258, 854-858. 12. HARDY, AND
J.
S. J. S., KURLAND, C. G., VOYNOW, P., MORA, G. (1969) Biochemistry 8, 2897-
2905. 13. OTAKA,
E.,
AND
KOBATA,
Genet. 162,259-268.
K.
(1978)
Mel
Gen.
OF YEAST
299
RIBOSOME
14. LOWRY, 0. H., ROSENBROUGH, N., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem 193,
265-275. 15. MICHEL, S., TRAUT, R. R., AND LEE, J. C. (1983) Mol. Gen. Genet. 191,251-256. 16. FENNER, C., TRAUT, R. R., MASON, D. T., AND WIKMAN-COFFELT, J. (1975) Anal B&hem. 63, 595-602. 17. Cox, R. A., AND HIRST, W. (1976) B&hem. J. 160, 505-519. 18. NAZAR,
R., YAGUCHI,
M., WILLICK,
C. F., AND ROY, C. (1979) Eur. 102,573-582.
G. E., ROLLIN,
J. B&hem.
19. REYES, R., VAZQUEZ, D., AND BALLESTA, J. P. G. (1978) B&him. Biophys. Acta 521, 229-234. 20. KRIJISWIJK, T., PLANTA, R. J., AND KROP, J. M. (1978) Biochim. Biophvs. Acta 517, 378-389. 21. ZINKER,
S., AND WARNER,
Chem. 251, 1799-1805.
J. R. (1976)
J. Biol