Reactivity of proteins in ribosomes from Saccharomyces cerevisiae with trypsin

ARCHIVES OF BICMZIEMISTRY AND BIOPHYSICS Vol. 224, No. 1, July 1, pp. 69-76, 1963

Reactivity

of Proteins in Ribosomes from Saccharomyces cerevisiae with Trypsin’ JOHN

Department of Biochemistry, Received

C. LEE University

December

AND

WILLIAM

T. HALEY,

JR?

of Texas Health Sciace Center, San Antonio, Texm 78284

7, 1982, and in revised

form

February

28, 1983

The susceptibility of 40s and 60s ribosomal subunits from Saccharmyces cerewisiae to digestion with varying concentrations of trypsin was studied by two-dimensional electrophoresis and quantitative measurements of the protein remaining with the ribosomal particles after trypsin treatment. Proteins from both subunits can be classified into three groups according to their rate of digestion by trypsin. These results are in good agreement with those obtained on the order of ribosomal assembly in vivo, i.e., proteins which are most susceptible to trypsin digestion have been shown to associate with the ribosomal particles at a relatively late stage of ribosome assembly.

Knowledge about the spatial arrangement of eucaryotic ribosomal proteins will be important in understanding the function and mechanism of action of the ribosome. One experimental approach to obtain information about the structural organization of the ribosome is the use of proteolytic enzymes to digest ribosomal protein in situ followed by analysis of the susceptibility of proteins to enzyme digestion. Ostner and Hultin (1) first studied digestion of rat liver ribosomes with either trypsin, chymotrypsin, or pronase and followed the disappearance of proteins by one-dimensional polyacrylamide-gel electrophoresis. They were able to classify ribosomal proteins according to their ease of digestion. Subsequently, Henkel et al (2) and Arpin et & (3) conducted similar studies except that two-dimensional polyacrylamide gels were used to analyze the proteins extracted from trypsin-treated ribosomal subunits. Although all proteins could be digested by trypsin, there were 1 This investigation the Robert A. Welch ‘Present address: tonio, Texas 78284.

differences in their sensitivity toward the enzyme. Proteins from both subunits were classified according to their trypsin sensitivity. Similar studies were conducted with Escherichia coli ribosomes (4-7). The E. coli ribosomal proteins were classified on the basis of their rate of digestion by trypsin. For the 30s subunits, the correlation between the susceptibility of the protein to tryspin digestion and its position in the assembly map (8) was satisfactory, i.e., the proteins removed from the ribosomal subunits first by trypsin appeared to be the last ones added to the ribosome in the reconstitution studies. The present report describes results obtained on trypsin digestion of 40s and 60s ribosomal subunits of Saccharomyces cerevisiae followed by electrophoretic analysis of the proteins extracted from the treated subunits on two-dimensional gels and quantitative measurements of the undigested proteins. The ribosomal proteins are classified according to their sensitivity toward trypsin. These results are compared to those obtained from studies on the in vivo assembly of ribosomal subunits (9).

was supported by a grant from Foundation (AQ 541 to JCL). San Antonio College, San An-

69

0003-9861/83 Copyright All rights

$3.00

0 1983 by Academic Press. Inc. of reproduction in any form reserved.

70

LEE AND HALEY

EFFECTS TABLE

OF

TRYPSIN

ON

YEAST

TABLE

I

SENSITIVITY OF 40s PROTEINS TRYPSIN DIGESTION

71

RIBOSOMES

TOWARD

II

CLASSIFICATION OF 40s AND 60s RIBOSOMAL SUBUNIT PROTEINS ACCORDING TO THE SENSITIVITY TOWARD TRYPSIN

P value Proteins

Time

of digestion

Degree of sensitivity

(min)

Protein

5

25

75

Sl

100

100

100

100

s4 S6 S7 SlO Sll s12 s13 s14 s17 S18 s19 s20 s21 s22 S24 S27 S27a S28 s31 S32 s33 s37

106 100 100 70 80 80 70 80 106 90 70 100 70 50 90 90 100 --a

80 100 90 65 50 80 60 70 70 90 50 106 65 30 -

80 100 70 65 40 72 50 10 0 90 100 65 90 90 80 -

60 90 70 65 40 70 50 0 0 80 40 100 70 10 80 80 -

100 80 100 90

20 30 90 40

0 15 50 20

0 10 50 10

10

Note. Ratio of trypsin to ribosome used in these experiments was 0.005 units/Aan. P = (Am of a protein normalized to that of Sl after digestion/A, of a protein normalized to that of Sl before digestion) X 100%. Average of four independent determinations.

“Not

determined. EXPERIMENTAL

40s

60s

I Sensitive (P G 30)

s14, 17, 22, 31, 32, 37

L4, ‘7, 8, 16, 17, 23, 26, 30, 32, 40, 41, 42

II Intermediate (p = 31-69)

SlO, 11, 13, 19, 21, 23, 33

LlO, 15, 18, 20, 21, 25, 29, 84, 36, 39, 46

Sl, 4, 6, 7, 12, 18, 20, 24, 27,27a

L2, 3, 5, 12, 19, %31,% 85,37,38

150

PROCEDURES

Motori&. Trypsin, treated with L-(l-tosylamido2-phenyl) ethylchloromethyl ketone to inhibit contaminant chymotryptic activity, was purchased from Worthington. pToluenesulfonyl-L-arginine methyl ester and soybean trypsin inhibitor were obtained

III

Resistant

(p = 70-100)

from Sigma Chemical Company. Before use, the enzymatic activity was determined with TAMEa as substrate (10). One unit of activity equals to the hydrolysis of 1 Nmol of TAME per minute at 25°C and pH 8.1 in the presence of 0.01 M calcium ion. The enzyme is approximately 50% as active in the ribosome buffer (see below for composition) as in the standard assay buffer. Yeast culture and ribosm preparation Sacchar~ myces cereuisiae (ATCC 22244) were grown and harvested in late exponential phase essentially as described (11). Ribosomes and subunits were isolated by zonal centrifugation as described (11). Trypsin digestion Purified ribosomal subunits in 50 mM Tris-HCl (pH 7.7), 10 mM MgClz, 10 mM @mercaptoethanol, and 50 mM NH,Cl were incubated with varying concentrations of trypsin-TPCK for different durations at 37°C. At the end of the incubation, the reaction was stopped by adding a concentrated solution of soybean trypsin inhibitor to give 1 mg of inhibitor per milligram of trypsin. The samples were incubated for an additional 5 min at 37°C and placed on ice. Protein analysis. Proteins were extracted from the

a Abbreviations used: TAME, p-toluenesulfonyl-Larginine methyl ester; TPCK, L-(1-tosylamido-2phenyl) ethylchloromethyl ketone.

FIG. 1. Two-dimensional polyacrylamide-gel electrophoresis analysis of 60s and 40s ribosomal proteins from Saccharomyces cerevisiae. Total proteins from each subunit were analyzed on twodimensional gels according to the procedure of Kaltschmidt and Wittmann (13). The direction of electrophoresis is from left to right in the first dimension and from top to bottom in the second dimension. The nomenclature of these ribosomal proteins is that proposed previously from this laboratory.

LEE

75

150

AND

HALEY

75

150

TIME OF DIGESTION,

75

150

MIN

FIG. 2. Rates of trypsin digestion of ribosomal proteins in situ. Ribosomal subunits were treated with 0.005 units of trypsin per A re, of ribosome at 3’7°C for the time indicated. Proteins remained associated with the ribosomal particles were analyzed on two-dimensional gels and quantitated as described under Experimental Procedures.

reaction mixture with 67% acetic acid and 0.1 M MgClz as previously described (12). The RNA pellets were reextracted and the combined protein extract was chromatographed on Sephadex G-25 in 1 M acetic acid. The protein-containing fractions were pooled and lyophilized to dryness. Proteins were dissolved in sample buffer and analyzed by two-dimensional polyacrylamide-gel electrophoresis basically as described by Kaltschmidt and Wittmann (13). except that a small-scale apparatus was used. The proteins were stained with amido black and numbered as described.’ To measure the quantitative distribution of the proteins, each protein spot was excised, eluted with 0.5 ml of 25% pyridine (14), and the optical density of the solution at 635 nm determined. To ensure uniformity, the relative amount of each protein was normalized to that of protein L5 and Sl for the 60.9 and 40s ribosomal proteins, respectively, for each gel. The relative amount of each protein was reproducible for different gels. The absorbance was proportional to the amount of proteins up to 20 pg. Variations among different experiments were about 10% in the four independent experiments. The intensity of some proteins on the two-dimensional gels was not sufficient to follow their digestion by trypsin accurately and therefore no data concerning these proteins are included. The amount of protein that remained after trypsin treatment is expressed by the P value (P value = [normalized amount

4 Michel, S., Traut, for publication.

R. R., and Lee, J. C., submitted

of protein remaining after digestion/normalized amount of protein before digestion] X 100%). RESULTS

Figure 1 shows a typical electrophoretic pattern of the basic proteins of the 40s and 60s ribosomal subunits of S. certiiae as separated by two-dimensional polyacrylamide-gel electrophoresis. The nomenclature of these ribosomal proteins follows that proposed previously from this laboratory.4 After trypsin-TPCK digestion of the 40s ribosomal subunit for ‘75 min at 27°C at an enzyme concentration of 0.005 units per AZ60of ribosome, most of the proteins were affected and either disappeared or became less visible. A few proteins (Sl, S6, S18, S20, S24, and S27) remained unaltered in intensity. Under similar digestion conditions, most 60s subunit proteins were affected; only proteins L5, L12, L19, L22, L33 remained unchanged. In both cases, a few new spots corresponding to tryptic peptides were observed on the two-dimensional gels. In order to obtain more information, 40s subunits were digested with trypsin-TPCK for various times, proteins remaining with the ribosome were analyzed by two-di-

EFFECTS

OF

TRYPSIN

mensional gel electrophoresis and their quantities measured. The data is summarized in Table I. The results represent the average of data from four independent experiments with different preparations of ribosomal subunits. The different sets of data were in good agreement with deviations of 10-1576.

TABLE SENSITIVITY

OF 60s PMTEINS

ON

YEAST

TABLE

Order assembly

Protein L2 L3 L4 L5 L7 L8 LlO L12 L15 L16 L17 L18 L19 L20 L21 L22 L23 L25 L26 L29 L30 L31 L32 L33 L34 L35 L36 L37 L38 L39 L40 L41 L42 L46 Nota Average Trypsin/ribosome,

150

80 80 65 196 95 90 95 199 90 85 90 95 166 90 95 166 166 f30 75 95 70 95 70 95 75 85 109 60 95 109 so 80 80 95

80 80 60 109 75 75 75 95 60 50 45 80 75 60 80 85 10 65 10 65 10 75 50 85 65 75 85 60 80 70 30 60 60 70

70 75 10 166 30 30 50 95 60 15 20 55 95 50 40 85 10 60 10 60 10 75 30 95 35 75 70 60 70 40 al 20 30 50

of four independent 0.095 units/A=.

determinations.

Trypsin (this

reactivity study)

Resistant Resistant Resistant Intermediate Resistant Intermediate Sensitive Sensitive Resistant Intermediate Sensitive Resistant Resistant Resistant

Late

SlO s20 s21 s31 S32 s33 S37

Intermediate Resistant Intermediate Sensitive Sensitive Intermediate Sensitive

(min)

75

Protein s4 S6 s7 Sll s12 s13 s14 s17 S18 s19 S22 S24 S27 S27a

TO TRYPSIN DIGESTION

5

of (9)

Early

III

of digestion

IV

COMPARISON BETWEEN ASSEMBLY STUDIES AND TRYPSIN DIGESTION OF 40s RIBOSOMAL SUBUNIT PROTEINS

P value Time

73

RIBOSOMES

On the basis of their sensitivity in situ to trypsin, these ribosomal proteins are classified into three groups (Table II). Within experimental errors, the relative rates of disappearance of Group I proteins appeared to be similar and the results of a few representative proteins are shown (Fig. 2). Two of the proteins (S17 and S31) were removed completely from the ribosomal subunits after 75 min of digestion; a third protein (S14) was degraded completely after 150 min. Some of the Group II proteins were digestable initially; what remained associated with the ribosome after the initial cleavage became refractory to trypsin digestion. Proteins SlO and S21 appeared to be readily modified by trypsin but were not degraded further after the initial rapid degradation. Visual inspection of the two-dimensional gel revealed that the electrophoretic mobilities of proteins SlO and S21 were slightly shifted. Based on the direction of shift, one could

74

LEE TABLE

AND

V

COMPARISON BETWEEN ASSEMBLY STUDIES, RNA BINDING, AND TRYPSIN DIGESTION OF THE 60s RIBOSOMAL SUBUNIT PROTEINS Order of assembly (9) Early

5.85 RNA binding (11) -

+ + -

Late

+ -

Protein

Trypsin digestion (this study)

L2 L3 L4 L5 LlO L12 L17 L18 L19 L20 L21 L22 L25 L26 L29 L31 L33 L34 L35 L37 L38 L39 L46

Resistant Resistant Sensitive Resistant Intermediate Resistant Sensitive Intermediate Resistant Intermediate Intermediate Resistant Intermediate Sensitive Intermediate Resistant Resistant Intermediate Resistant Resistant Resistant Intermediate Intermediate

L7 L8 L15 L16 L23 L30 L32 L36 L40 L41 L42

Sensitive Sensitive Intermediate Sensitive Sensitive Sensitive Sensitive Intermediate Sensitive Sensitive Sensitive

infer that a basic and an acidic oligopeptide was removed from protein SlO and S21, respectively. Four proteins from the 40s subunit were entirely resistant to trypsin digestion (Sl, S6, S20, S27, Group III). The rates of digestion of a few representative 40s proteins in each class are shown in Fig. 2. Control experiments, in which isolated 40s proteins were treated with trypsin under identical conditions, showed that all proteins were extensively digested by trypsin though the extent of degradation varied from one protein to another (data not shown).

HALEY

Table III summarizes the trypsin digestion data for the 60s subunits. The data represent the average of data from four independent experiments with different preparations of ribosomes. The agreement was even better for the different sets of data with variations of 5-10s. On the basis of their sensitivity in situ to trypsin, the 60s proteins are classified into three groups (Table II). The rates of digestion of several representative 60s proteins in each group is shown in Fig. 2. Visual inspection of the gel indicated that proteins L25 and L38 appeared to have lost a basic oligopeptide fragment(s) from one or both of the termini of the proteins by trypsin digestion, but the remainder of the protein molecule was resistant to further enzymatic digestion. The loss of the fragment would result in a slight shift in the electrophoretic mobilities of the protein on the two-dimensional polyacrylamide gel. Four proteins from the 60s subunit were completely resistant to trypsin (L5, L12, L19, L33, Group III). In order to determine whether the differences detected in the sensitivities of the various ribosomal proteins in the 60s subunits were due to an intrinsic property of these proteins, or to their organization in the ribosomal subunits, isolated free ribosomal 60s proteins were treated with trypsin under identical conditions and analyzed by two-dimensional gel electrophoresis. All of the free proteins were extensively digested, though the extent of degradation varied from protein to protein (data not shown). DISCUSSION

The present study demonstrates the application of trypsin digestion of ribosome in situ and two-dimensional polyacrylamide-gel electrophoresis to the investigation of ribosomal protein reactivities of a low eucaryote. According to their sensitivities towards trypsin, yeast ribosomal proteins from both subunits have been classified into three classes: I (sensitive), II (intermediate), and III (resistant). A comparison between the susceptibility of the ribosomal proteins toward tryp-

EFFECTS OF TRYPSIN ON YEAST RIBOSOMES

75

TABLEVI COMPARISON

OF RIBOSOMAL AND

PROTEINS

LABELED

WITH

AmioAcn-tRNA

DERIVATIVES

THEIR SENSITIVITY TOWARD TRYPSIN

Ribosomal sites (20) A-site PNPC-pbe-tRNA

P-site NIA-phe-tTNA

PNPC-phe-tRNA

NIA-phe-tRNA

L2 L4/L6 L7 L9 L17/L18 L19/L20

L4/L6

L19/L20 L22/L23 L26

L29 L36 L42 L43

L36 L38 L39

L36

L43

sin and the order of assembly of these proteins on both ribosomal subunits (9) is shown (Tables IV and V). Of the 40s ribosomal proteins, majority (80%) of our Group III proteins is classified as “early” assembled proteins by Kruiswijk, et al. (9) whereas 50% of the Group I protein is classified as “late”. Hence, the correlation between the susceptibility of a specific protein in the ribisomal subunit to trypsin and its order of assembly on the subunit appears to be good. Table V also includes a list of proteins which have been demonstrated previously to form specific complexes with 5.8s RNA (11). All these proteins are relatively insensitive to trypsin digestion except L23 and L30 which are sensitive to trypsin. The reason for this difference is unclear, and the scarcity of data on eucaryotic ribosomes makes it difficult to resolve these apparent differences at the present. In the case of E. coli, all 30s ribosomal proteins which are required for assembly and those that form specific complexes with the 16s RNA in vitro are indeed relatively slow in reaction with trypsin (6). Several of the yeast proteins, which as-

L36

L43

Trypsin digestion (this study) Resistant Sensitive Sensitive Undefined Sensitive/Intermediate Resistant/Intermediate Resistant/Sensitive Sensitive Intermediate Intermediate Resistant Intermediate Sensitive Undefined

semble late during ribosome maturation, can undergo post-translational phosphorylation or methylation (15-19). Our data indicate that these modified proteins or regions of these proteins are quite accessible to trypsin (SlO, S31, S32, L15, L30, and L41). Of particular interest is protein SlO which appears to be homologous to S6 in higher eucaryotes. The degree of phosphorylation of S6 is influenced by the physiological conditions of the tissues. Even though SlO is relatively resistant to tryspin, it contains a basic amino acid sequence that is very sensitive to trypsin. Perhaps this is the region of the protein which is phosphorylated. As shown in Table VI, majority (88%) of the proteins ascribed as in the vicinity of the ribosomal A-site by affinity labeling (20) are relatively sensitive to trypsin digestion with P values less than 70. By comparison, 79% of the large subunit proteins have P values less than 70. With the exception of protein L19,100% of the proteins ascribed as in the neighborhood of ribosomal P-site by affinity labeling (20) are also relatively sensitive to trypsin. In conclusion, the kinetics of disappear-

76

LEE

AND

ante of proteins from yeast ribosomal subunits as a result of trypsin digestion have been studied. These proteins have been classified into three groups based on their sensitivity to trypsin. In general, the data of trypsin digestion in situ are in good agreement with data obtained by several other experimental approaches.

HALEY

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