Differences in the distribution of phosphate content in the ribosomal subunit proteins of free and membrane-bound ribosomes from normal and regenerating rat liver

62

Biochimica etBiophysica Acta, 656 (1981) 62-68

Elsevier/North-HollandBiomedicalPress BBA 99954 DIFFERENCES IN THE DISTRIBUTION OF PHOSPHATE CONTENT IN THE RIBOSOMAL SUBUNIT PROTEINS OF FREE AND MEMBRANE.BOUND RIBOSOMES FROM NORMAL AND REGENERATING RAT LIVER DAVID P. RINGER, DONALD E. KIZER and ROBERT L. KING, Jr. The Samuel Roberts Noble Foundation, Inc., Route 1, Ardmore, OK 73401 (U.S.A.)

(Receded June 15th, 1981)

Key words: Ribosomal protein; Ribosome; Regeneration; Phosphate content; Protein phosphorylation; (Rat Hver)

Proteins of membrane-bound ribosomes from normal liver contained 60-70% more phosphate than did proteins from free ribosomes. This difference was not a reflection of the phosphate contents of respective 40 S subunits. Instead, it was owing to the presence of high levels of phosphorylated proteins in the 60 S subunits, i.e., phosphate contents equal to or greater than those for 40 S subunits. The proteins of membrane-bound 60 S subunits contained twice the phosphate as free 60 S subunits. In regenerating rat liver, membrane-bound ribosomes had increased phosphate in the proteins of the 40 S subunits and decreased phosphate in proteins of the 60 S subunit when compared to controls from normal rat liver. No significant changes occurred in the proteins of free ribosomes from regenerating rat liver. These findings are discussed with respect to (a) the importance of assessing total phosphate contents of proteins in the study of ribosomal protein phosphorylation, and (b) the possible involvement of ribosomal protein phosphorylation in the segregation of ribosomes into free and membrane-bound populations and the regulation of these distributions to meet changes in the translational demands of the cell.

Introduction Phosphorylation of ribosomal proteins has been observed in vivo in rat liver [10-13], rabbit reticulocytes [14-17], yeast [18-20], brine shrimp [21], plant [22], in situ in a number of cell culture systems [6,7,10,23-31], but not in bacteria [32]. In spite of numerous observations of altered patterns of ribosomal protein phosphorylation accompanying rapid cellular growth [5,27,33], the administration of hormones [3,4,12,23,34] or cyclic AMP [5,16,24,34], presence of inhibitors of protein synthesis [11,14, 18], variation of cell culture nutritional conditions [20,25], virus infection [10,28] or presence of cytotoxic agents [8,13], no specific functional significance has yet been attributed to this class of ribosomal protein modification. Most of the information concerning in vivo ribosomal protein phosphorylation has relied on the

extent to which inorganic [32P]phosphate was incorporated during short pulses under the imposed conditions of rapid cell proliferation or xenobiotic action. Possible shortcomings associated with this approach include (a) failure to measure total ribosomal protein phosphate, (b) an inability to distinguish between new sites of phosphorylation and phosphate turnover at pre-existing sites, (c) the possibility that conditions or agents that stimulate inorganic [32p]phosphate incorporation are unique from homeostatic mechanisms that regulate protein phosphorylation. Removal of such limitations may be necessary before an understanding of the functional significance of ribosomal protein phosphorylation is possible. The direct quantitation of the total phosphate content of ribosomal proteins instead of, or in addition to, estimates of extent of phosphorylation following short pulses of inorganic [32P]phosphate, may lead to a better understanding of ribosomal protein phos-

0005-2787/81/0000-0000/$02.50 © 1981 Elsevier/North-HollandBiomedicalPress

63 phorylation. In this study, a colorimetric assay for phosphate with nanomole sensitivity was used to measure the phosphate content of ribosomal protein in 40 S and 60 S subunits that were isolated from free and membrane-bound ribosomes of normal and regenerating rat liver. Our findings indicate that membrane-bound ribosomes from normal rat liver has significantly greater content of phosphorylated protein than free ribosomes, and that significant changes in phosphate distributions occurred in membrane-bound ribosomes during liver regeneration. Materials and Methods

Materials. Reagents for the phosphate assay included sterox (Harleco, Philadelphia, PA), malachite green (MCB Manufacturing Chemists, Inc., Norwood, OH) and ammonium molybdate (J.T. Baker Chemical Co., Arlington, TX). Alkaline phosphatase (EC 3.1.3.1), acid phosphates (EC 3.1.3.2) and bovine serum albumin were obtained from Sigma Chemical Co., St. Louis, MO. Animals. The source of rat liver ribosomes used in these studies was female Holtzman rats weighing 125-175 g. They were maintained from birth on 12-h light and dark cycles and were fed ad libitum Teklad Rat and Mouse Diet (Teklad Mills, Winfield, IA). Ribosomes from regenerating liver were isolated 24 h after partial hepatectomy [35]. All animals were fasted for 18 h prior to killing by cervical dislocation. In each experiment ribosomes were isolated from a pool of eight rat livers. Preparation of ribosomal subunits from free and membrane-bound ribosomes. Free and membranebound ribosomes were isolated from the post-nuclear supernatants of livers as described by Venkatesan and Steele [36] except that free ribosomes also were deoxycholate-treated before final pelleting. As previously shown [36], this technique employs differential centrifugation of liver homogenate to obtain a nearly quantitative recovery (90%) of free and membrane-bound ribosomes from rat liver with minimal cross-contamination (4-14%). Pelleted ribosomes were resuspended in 20mM Tris-HC1 (pH 7.5)/150 mM NH4C1/4mM MgCI2/1 mM dithiothreitol, and then incubated for 20 min at 37°C in the presence of 1 mM ATP/0.4mM GTP/0.2mM puromycin to facilitate the removal of nascent peptide and endog-

enous mRNA [37]. Following puromycin treatment, KC1 was added to give a final monovalent cation concentration of 0.5 M and ribosomes were incubated for 10 min at 37°C. After KC1 treatment, ribosomes were layered onto 12-38% sucrose gradients containing 20 mM Tris-HC1 (pH 7.5)/300 mM KCI/3 mM MgC12/ 1.0 mM dithiothreitol. The 40 S and 60 S subunits were isolated from their respective regions of the gradients following overnight centrifugation (660 000 g . h) in a Beckman SW27 rotor as described by Falvey and Staehelin [38]. Pooled 40 S and 60 S subunits were pelleted from their respective gradient fractions by centrifugation in a Beckman 40 rotor (1 408 000 g. h) and then resuspended in 20 mM Tris-HC1 (pH 7.5)/100 mM NH4C1/2 mM MgC12/ 1 mM dithiothreitol to give a final subunit concentration of 40-100 A26o units/ml. The purity of the 40 S and 60 S subunits was routinely determined by analysis of their rRNA contents on sucrose density gradients. In a typical analysis, 0.5-2.0A26o units of subunits were de-proteinized by either treatment with 0.5% sodium dodecyl sulfate [38] or extraction by phenol/sodium dodecyl sulfate [39], and the rRNAs were separated by centrifugation (Beckman SW 50.1 rotor, 390 000 g. h) into 12-38% linear sucrose gradients containing 20 mM Tris-HC1 (pH 7.5)/100 mM NH4C1/1 mM MgC12. All 40 S subunit pools were clear of 28 S RNA, indicating no 60 S subunit contamination. The 60 S subunit pools from free and membrane-bound ribosomes were found to contain low levels (7 +- 1%) of 18 S RNA from 40 S subunits. To remove co-sedimenting ferritin from the subunits, especially 60 S subunits, they were precipitated by the addition of MgCI~ to a final concentration of 50 mM, the precipitates collected by centrifugation and pellets washed by resuspending in 10 mM MgC12 and again centrifugally pelleted [40]. Although these pellets could be stored at -80°C without detectable change in phosphorylation of ribosomal proteins, they were routinely processed immediately. Preparation of ribosomal proteins. Ribosomal subunit pellets were resuspended in 20 mM Tris-HC1 (pH 7.5)/100 mM NH4CI/2 mM MgC12/3 mM dithiothreitol and ribosomal proteins extracted by the 100 mM MGC12/67% glacial acetic acid method of Sherton and Wool [37]. Following centrifugal removal of precipitated rRNA, ribosomal proteins were precipitated by the addition of 5 vols. acetone and protein precip-

64 itates were collected by centrifugation [41]. The isolated ribosomal proteins were next subjected to electrophoresis through a gel plug to obtain a nearly complete removal of rRNA contaminants. For this, proteins were resuspended in 1 M acetic acid/5 mM dithiothreitol/10% sucrose and applied to disc gel electrophoresis columns containing 7.6% polyacrylamide gel plugs (1 × 1.5 cm). Attached to the bottom of each column were bags constructed of dialysis tubing (Spectrapor 3, Spectrum Medical Industries, Inc., Los Angeles, CA) which extended down into the lower buffer chamber of the electrophoresis unit and were filled with electrophoresis buffer. Electrophoresis was at pH 3.2 in 0.9 M acetic acid [42] for 4 h at 6 mA/gel. Dialysis bags were removed from gel columns and dialyzed overnight (1 : 200) against 1 mM dithiothreitol to remove inorganic phosphate contaminants. One-dimensional polyacrylamide disc gel electrophoretic comparisons of 40 S and 60 S subunit proteins before and after electrophoresis through gel plugs were conducted under two conditions, i.e., at pH 3.2 [42] and in sodium dodecyl sulfate [43]. No significant changes in protein electrophoretic profiles appeared (data not shown). These preparations of ribosomal proteins were next examined for protein content by the method of Lowry et al. [44] with bovine serum albumin as the standard, and finally for phosphate content (as given below). Colorimetric determination of phosphate. Quantitation of the phosphate content of ribosomal proteins was performed by the micro-assay of Hess and Derr [45]. A typical determination consisted of exhaustive digestion of 100-200/ag of dried ribosomal protein for 30 min at 180-190°C in sulfuric and perchloric acids, detection of inorganic phosphate by the ammonium molybdate-malachite greensterox reagent, and extent of color development measured at 660 nm by a Gilford 250 spectrophotometer equipped with fused quartz microcuvettes. Simultaneous work-up of inorganic phosphate standards supplemented with 100-200/ag of bovine serum albumin were used to generate standard curves ranging from 0.5 to 4.0 nmol phosphate. The working portion of the curve was generally 0.5 to 1.5 nmol and determinations were routinely performed in duplicate.

Tests for non-phosphoprotein sources o f phosphate in ribosomal protein preparation. Tests used to

detect phosphate contaminants in our preparations of ribosomal protein were similar to those described by Bitte and Kabat [46]. To estimate contamination by RNA, an aliquot containing 200/~g of ribosomal protein was precipitated by cold 10% trichloroacetic acid. The pellet was resuspended and heated in 10% trichloroacetic acid at 90°C for 20 min and cooled, and the trichloroacetic acid precipitate was re-pelleted by centrifugation. The amount of phosphate released in the supernatant by the hot-trichloroacetic acid treatment was then determined by colorimetric assay. The quantity of contaminating phospholipid was estimated by colorimetric determinations of phosphate content before and after lipid extraction of 100 ~g dry ribosomal protein by chloroform/methanol ( 2 : 1 ) . Contaminating inorganic phosphate was eliminated as a possible source of phosphate by dialysis of protein against deionized water (1 : 200 overnight) prior to determination of phosphate content. Further evidence for the covalent nature of the remaining bound phosphate was obtained in studies employing alkaline or acid phosphatase to catalyze hydrolysis of the phosphomonoester bond linking phosphate to ribosomal proteins. Reaction conditions consisted of incubating 500/ag ribosomal protein for 2 - 6 h at 37°C in a final volume of 1.0 ml which contained either 10 mM Tris-HC1 (pH 8.0)/1 mM ZnC1J 100/ag of alkaline phosphatase, or 10mM sodium malate (pH 5.0)/500/ag of acid phosphatase. Following incubation, samples were dialyzed at 4°C overnight against deionized water (1 : 200) and the loss of phosphate determined from colorimetric assay of phosphate content. Results

Phosphate content of rat liver ribosomal proteins The phosphate content of proteins isolated from the free and membrane-bound ribosomes of rat liver is shown in Table I. Protein from membrane-bound ribosomes possessed significantly more phosphate than protein from free ribosomes. Since free and membrane-bound ribosomes constitute 40 and 60%, respectively, of the total cytoplasmic ribosomes in rat liver [36,47], the sum of the weighted averages for the free and membrane-bound ribosomes resulted in a value for the phosphate content for total cytoplasmic

65 TABLE I A COMPARISONOF THE PHOSPHATECONTENT OF RIBOSOMALPROTEIN FROM RAT LIVER FREE AND MEMBRANEBOUND RIBOSOMESWITH VALUES PREVIOUSLYREPORTED IN THE LITERATURE FOR RIBOSOMALPROTEIN Ribosome source

Ribosome population

Phosphate ribosome a

Rat liver Rat liver Rat liver

free membrane-bound cytoplasmic

15.0 ± 0.7 b 24.9 ± 2.1 b 20.9 ± 0.8 c

Rat liver Rabbit reticulocyte Sarcoma 180 cell

cytoplasmic cytoplasmic cytoplasmic

33- 37 14.2 25

Ref.

1,4 45 45

a Calculated on the basis that the eukaryotic ribosome contains 2.15 • 106 daltons of ribosomal protein [54 ]. b Values are mean ± S.E. and were obtained from Table II by summation of data for the respective 40 S and 60 S subunits. c Mean ± S.E. resulting from addition of appropriately weighted values for free and membrane-bound ribosomes (see text for further explanation).

ribosomes, i.e., 20.9 -+ 0.8 phosphates/ribosome. This value may be compared with values (Table I) that were previously reported in the literature for the phosphate content" of proteins from ribosomes representing the total cytoplasmic ribosomal pools. In normal rat liver [1,4] the phosphate levels of previous studies were almost 2-fold greater than our values. Since detergent and high-salt treatment of ribosomes is required to remove nonspecifically bound cellular proteins as well as cytoplasmic translation factors [46,48], some of which are known to be phosphorylated [14,49], failure to do so in the earlier studies probably accounts for their higher estimates of phosphate content. The value of approx. 14 phosphoryl groups in the proteins of the rabbit reticulocyte ribosome was obtained when the preparation included detergent and high-salt concentration treatments. This value for reticulocyte ribosomes, a predominantly free ribosome population [50], was similar to our value for free ribosomes from rat liver. Sarcoma 180 ribosomes contained higher phosphate levels in their proteins but also were reported to be slightly contaminated with non-ribosomal proteins [46]. While we considered these data to be colasistent with our findings that highly purified rat liver ribosomes had approx. 21 phosphoryl groups bound to their proteins, we also examined the possibility that our phosphate values may reflect the presence of nonphosphoprotein contaminants. Tests for contamination by RNA, phospholipids and inorganic phosphate

were conducted (see Materials and Methods). Hot trichloroacetic acid treatment of protein samples resulted in 7.5 ---2.3% of their total phosphate con. tents to be lost, indicating a detectable but tolerably low level of RNA contamination. No significant change in total phosphate content was found following extended dialysis (48 h) or organic solvent extraction, indicating no contamination by phospholipids or inorganic phosphate. Furthermore, treatment of ribosomal proteins with either alkaline or acid phosphatase caused at least 80% of total phosphate to become dialyzable. This suggests that most, if not all, bound phosphate was covalently linked by phosphomonoester bonds to ribosomal proteins. Failure to observe 100% release of bound phosphate may be owing to an inaccessibility of some phosphoryl groups following extraction and subsequent denaturation of ribosomal proteins.

Phosphate content of proteins from 40 S and 60 S subunits of free and membrane-bound ribosomes When the phosphate content of proteins from free and membrane-bound ribosomes was viewed at the subunit level (Table II), two important aspects of their phosphate distributions were revealed. Firstly, the proteins of the 60 S ribosomal subunits from both free and membrane-bound ribosomes contained amounts of phosphate equal to or greater than that of the corresponding 40 S ribosomal subunits. These high levels of phosphate in 60 S subunit protein have

66 TABLE II

TABLE III

PHOSPHATE CONTENT OF 40 S AND 60 S RIBOSOMAL SUBUNIT PROTEIN FROM FREE AND MEMBRANEBOUND RIBOSOMES

CHANGES IN PHOSPHATE CONTENT OF PROTEIN FROM RIBOSOMAL SUBUNITS OF RAT LIVER 24 h AFTER PARTIAL HEPATECTOMY

Protein source

Protein source

Free ribosomes Membrane-bound ribosomes

Phosphate content (phosphate/subunit) 40 S

60 S

6.6 ± 1.7 a 5.9 ± 0.8

8.4 ± 1.2 19.0 ± 5.6 b

a Values reported as mean ± S.E. for five experiments. b Value for membrane-bound subunit different from free subunit value at P < 0.05. never before been reported [46,48]. Secondly, the phosphate content of the membrane-bound 60 S subunit is 2-fold greater than that of 60 S subunits from free ribosomes. In contrast, the phosphate contents for the respective 40 S subunits were not significantly different. Thus, the difference observed in phosphate content between membrane-bound and free ribosomes was predominantly a reflection of differences between their respective 60 S subunits.

Changes in phosphate content of proteins extracted from ribosomal subunits of regenerating rat liver The functional significance of ribosomal protein phosphorylation is as yet undetermined. Searches for functional significance have usually employed examinations o f ribosomes from the livers o f rats treated to cause departure from normal physiological conditions. We chose to compare our phosphate distributions for ribosomal proteins from normal rat liver with those for ribosomes isolated from 24-h regenerating rat liver. As shown in Table III, relative to normal rat liver distributions, membrane-bound ribosomal subunits isolated from regenerating rat liver showed significant departures, i.e., 40 S subunits had a 50% increase in phosphate content, while 60 S subunits decreased to approximately one-half its normal value. Phosphate contents of proteins from free ribosomal subunits o f regenerating liver did not show statistically significant departures ( P < 0 . 0 5 ) from the values for normal rat liver although the 60 S subunit values suggested a strong tendency toward an increase in phosphate content. Increase in the phosphate content of 40 S subunit proteins o f regenerating liver has

Free ribosomes Membrane-bound ribosomes

Regenerating/normal a 40 S

60 S

1.02 ± 0.03 b

1.63 ± 0.29

1.53 ± 0.13 c

0.42 ± 0.03 c

a Ratios of the phosphates/subunit for ribosomal subunits from regenerating and normal rat liver. b Values are mean ± S.E. for four experiments. c Value significantly different from 1.00 at P < 0.05.

previously been reported in studies examining [32p]. phosphate incorporation [5]; however, due to a lack of significant labeling in 60 S subunit proteins, significant change in the phosphate content of 60 S subunit proteins has not previously been reported.

Discussion Free and membrane-bound ribosomes were prepared from the livers of rats and purified forms of their 40 S and 60 S subunits, i.e., detergent and highsalt-treated, isolated from sucrose density gradients. Proteins were extracted from the ribosomal subunits and examined for their phosphate content by colorimetric assay with the following results: (a) the proteins of the membrane-bound and free 40 S ribosomal subunits contained similar amounts of phosphate; (b) the proteins of both membrane-bound and free 60 S subunits contained high levels of phosphate, i.e., equal to or greater than that of the 40 S ribosomal subunits; and (c), the 60 S subunits of membrane-bound ribosomes contained more than twice the phosphate level found in the proteins of free 60 S subunits. Hence, the phosphate content of ribosomal proteins o f membrane-bound ribosomes was found to be significantly greater (1.67-fold) than that found in free ribosomes. The greater than 2-fold higher phosphate content of 60 S subunit proteins from membranebound ribosomes suggests that there may be structural basis for distinguishing between membrane-bound and free ribosomes. Whether such a structural differ-

67 ence may serve as a basis for functional differences between free and membrane-bound ribosomes poses an interesting possibility for ribosomal protein phosphorylation. The presence of high levels of phosphate in the 60 S subunits of free and membrane-bound ribosomes has not previously been reported. This may reflect a lack of rapid inorganic [32P]phosphate incorporation into 60 S subunit proteins relative to the well-studied incorporation into the proteins of the 40 S subunits [3,5,11,17,27,51]. It remains to be determined whether differences between phosphate distributions as assessed by inorganic [a2P]phosphate incorporation or by total phosphate content determinations may reflect (a) different mechanisms of phosphorylation utilizing separate phosphate pools, or (b) merely different rates of phosphate turnover during the short periods routinely used for incorporating inorganic [32Plphosphate. Proteins from free and membrane-bound ribosomes of 24-h regenerating rat liver were examined for phosphate content and compared to values for normal liver. Little or no significant change was found in the phosphate content of the subunit proteins from free ribosomes, while significant increases were observed in the phosphate content of subunit proteins from membrane-bound ribosomes. The failure of 40 S subunits from free ribosomes to show changes in phosphate content raises the interesting question of whether the increases in inorganic [32p]. phosphate incorporation previously observed in cytoplasmic 40 S subunits [5] following partial hepatectomy may primarily occur in 40 S subunits from membrane-bound ribosomes. The shift to lower phosphate content in the 60 S subunit proteins of membrane-bound ribosomes from regenerating rat liver may be another indication of an involvement of phosphorylation of 60 S subunits with the distribution of ribosomes between free and bound states. Loeb and Yeung [52] have indicated that a shift in the distribution of cytoplasmic ribosomes toward increased free ribosomes occurs in regenerating rat liver as it shifts the balance of protein synthesis to favor synthesis of cellular proteins over those for export. In addition to the phosphorylation differences reported here, at the levels of free versus membranebound ribosomes, differences have been reported at a number of other levels of ribosomal organization. The

40 S subunit undergoes large increases in inorganic [a2P]phosphate incorporated by protein S-6 following partial hepatectomy [5,51 ], and differences have been reported in phosphate distribution between the ribosomal proteins of monosomes and polyribosomes [15,53]. In light of this, it is quite possible that the basic patterns of phosphate content reported here do not reflect a single aspect of ribosome function during translation. In order to correlate change(s) in phosphorylation patterns more specifically with respect to function(s), it will be necessary to extend these studies to the level of individual ribosomal protein analysis, e.g., by two-dimensional slab-gel electrophoresis, and monitor changes in their phosphorylation with respect to both phosphate content and change in inorganic [32P]phosphate incorporation. By monitoring phosphorylation changes in individual ribosomal proteins from normal and physiologically perturbed animals with respect to these two distinct aspects of phosphorylation, a more informative basis for understanding the structure-function relationships of ribosomal protein phosphorylation may follow.

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