evolution of proteins in mammalian cytoplasmic and mitochondrial ribosomes

J Mol Evol (1986) 24:110-117

Journal of Molecular Evolution ~) Spdnger-Verlag New York Inc. 1986

Evolution of Proteins in Mammalian Cytoplasmic and Mitochondrial Ribosomes*

Salvatore F. Pietromonaco,** Robert A. Hessler, and Thomas W. O'Brien

Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Box J-245, J, Hillis Miller Health Center, Gainesville, Florida 32610

Summary. The proteins of cytoplasmic and mitochondrial ribosomes from the cow and the rat were analyzed by co-electrophoresis in two dimensional polyacrylamide gels to determine their relative evolutionary rates. In a pairwise comparison of individual ribosomal proteins (r-proteins) from the cow and the rat, over 85% of the cytoplasmic r-proteins have conserved electrophoretic properties in this system, while only 15% of the proteins of mitochondrial ribosomes from these animals fell into this category. These values predict that mammalian mitochondrial r-proteins are evolving about 13 times more rapidly than cytoplasmic r-proteins. Based on actual evolutionary rates for representative cytoplasmic r-proteins, this mitochondrial r-protein evolutionary rate corresponds to an amino acid substitution rate of 40 • 10-~0 per site per year, placing mitochondrial r-proteins in the category of rapidly evolving proteins. The mitochondrial r-proteins are apparently evolving at a rate comparable to that of the mitochondrial rRNA, suggesting that functional constraints act more or less equally on both kinds

Offprint requests to: Thomas W. O'Brien

* Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1-5, 1986

** Present address: Department of Cell Biology, Yale University, New Haven, Connecticut 06510 Abbreviations; mitoribosome, mitochondrial ribosome; cytori- bosome, cytoplasmic ribosome; r-protein, ribosomal protein; mit, mitochondrial; cyt, cytoplasmic

of molecules in the ribosome. It is significant that mammalian mitochondrial r-proteins are evolving more rapidly than cytoplasmic r-proteins in the same cell, since both sets of r-proteins are encoded bY nuclear genes. Such a difference in evolutionary rates implies that the functional constraints operating on ribosomes are somewhat relaxed for mitochondrial ribosomes.

Key words: Electrophoretic analysis -- Ribosomal proteins -- rRNA -- Mitochondrial ribosomes Cytoplasmic ribosomes -- Mitochondria

Introduction

The ribosomes in mammalian mitochondria have unusually small RNAs and a correspondingly higher protein content than other ribosomes. Representa- tive of mammalian mitochondrial ribosomes (mitoribosomes), the bovine mitoribosome contains as many as 85 different proteins, all of which are products of nuclear genes. These proteins are synthesized on cytoplasmic ribosomes (Schieber and O'Brie~a 1985) and must therefore be imported by mitochondria for assembly with the mitochondriaUy encoded rRNA. Sequence analysis of the mitoribosomal RNAs reveals that they are evolving significantly more rapidly than the corresponding RNA in cytoplasmic ribosomes (cytoribosomes). It is therefore of interest to determine evolutionarY rates for the mitoribosomal proteins which, al-

though they a re n u c l e a r gene p r o d u c t s , m u s t a s s e m - ble wi th a r a p i d l y e v o l v i n g R N A . T h e p r o t e i n s in

m a m m a l i a n m i t o c h o n d r i a l r i b o s o m e s a p p e a r to b e Changing m o r e r a p i d l y t h a n t h o s e f r o m c y t o p l a s m i c r ibosomes , g iv ing r ise to a l t e r e d e l e c t r o p h o r e t i e m o - bil i t ies o f the p r o t e i n s in in t e r spec i f i c c o m p a r i s o n s (Mat thews et al. 1978). In t he a b s e n c e o f a m i n o a c i d Sequence i n f o r m a t i o n , t he se d i f fe rences in m o b i l i - ties are usefu l in a s ses s ing the r e l a t e d n e s s o f h o - mologous p r o t e i n s in d i f fe ren t species . W e u n d e r - took the p r e sen t s tudy to iden t i fy i n d i v i d u a l p r o t e i n s o f ra t a n d c o w m i t o c h o n d r i a l r i b o s o m e s h a v i n g altered e l e c t r o p h o r e t i c p r o p e r t i e s in o r d e r to def ine the na tu re a n d ex t en t o f v a r i a t i o n o f p r o t e i n s in m a m m a l i a n m i t o r i b o s o m e s . F u r t h e r m o r e , we h a v e also Conduc ted a s i m i l a r e l e c t r o p h o r e t i c ana lys i s o f bovine a n d ra t c y t o p l a s m i c r - p r o t e i n s in o r d e r to establ ish the ra te o f c h a n g e o f m i t o r i b o s o m a l p r o - teins r e l a t ive to t h a t o f p r o t e i n s in c y t o p l a s m i c r i - bosomes .

Exl*erimentai Procedures

Preparation of Ribosomes

Mitochondrial and cytoplasmic ribosomes were prepared from bovine and rat livers as described previously (Matthews et at. ~982). Mitoribosomal subunits were derived from 55S ribosomes ~Y centrifugation in 10--30% linear sucrose gradients containing ~00 mM KCI, 5 ram MgC12, 10 mM Tris-HCl, pPI 7.5, and 5 ~ m 2-mercaptoethanol. Subribosomal particles were coneen- �9 ,~tte~l hy eentrifugation for 15 h at 230,000 x g in a Beckman TY!~ 65 rotor, resuspended in 25 mM KCi, 2.5 mM MgCI2, 5 r~M 2"mercaptoethanol, and 10 mM triethanolamine, pH 7.5, i iO d Stored at - 70"C until needed. Cytoribosomal subunits were

red from 80S ribosomes by sucrose density centrifugation in mM KCi, 5 mM MgC12, 5 mM 2-mereaptoethanol, and 20

haM triethanolamine, pH 7.5, and the subunits were concentrated arid Stored as described above.

l~xtraction and Radioactive Labeling of Ribosomal Proteins

~bos.omal proteins were extracted using 9 M urea, 3 M LiC1 as ~.~Senbed previously (Matthews et al. 1982). Proteins were la- ~ d by reduetive methylation using cyanoborohydride and ,~,~|f~ (Jentoft and Dearborn 1979). Two nmol of 'to~~ (42 Ci/mol, New England Nuclear) were added 8 ~v/~g of ribosomal proteins in a volume of 24 ~1 containing . ; ~ urea, 3 M LiC1, 20 mM NaCNBH~, and 20 mM KH2 PO4, v~ 7.2. The reaction proceeded with constant stirring for two -uurs at room temperature, and the mixture was then dialyzed against 9 M urea, 60 mM potassium acetate, pH 6.7, and 0.01% anainoethanethiol.

7"~Vo Dimensional Polyacrylamide Gel Electrophoresis

aRiboso.mal proteins were separated by two-dimensional poly- crYlamlde el Matthews et al 1982 S- . g electrophoresis as described ( . ). etaaratlon in the first dimension was in 4.6% polyacrylamide

gels containing 9 M urea and 60 mM potassium acetate, pH 4.3,

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and the second dimension in a 10% polyacrylamide slab gel con- mining 5 M urea, 0.5% sodium dodecyl sulfate, and 100 mM sodium phosphate, pH 7.2. After electrophoresis, the gels were stained with Coomassie brilliant blue R and photographed. The gels were prepared for fluorography (Chamberlain 1974; Laskey and Mills 1975) and ink containing [J4C]amino acids was spotted on the edges of the dried gels before they were placed in contact with pre-flashed Kodak XAR-5 X-ray film. The radiolabeled ribosomal proteins were located relative to the stained proteins by superposition of the fluorogram on the dried gel using the reference marks.

Pairwise Analysis of Ribosomal Proteins

We used a co.eleetrophoretic procedure to compare the mobilities of rat and cow proteins in two-dimensional polyacrylamide gels. To obtain an accurate relative posit/oning of the cytoplasmic ribosomal proteins from these two sources, trace quantities (5- 10 ~g total protein) of rat proteins (from either subunit) were labeled with [~4C]formaldehyde by reduetive methylation and co- electrophoresed with a larger (about 200 ug total protein) stainable quantity of proteins from the corresponding subunit of bovine cytoplasmic ribosomes. After electrophoresis, staining and fluorography, the patterns of stained (bovine) and radioactive (rat) proteins were compared. The relative electrophoretie mobilities of these proteins were also determined in reciprocal experiments, using radiolabeled cow proteins and stainable amounts of rat proteins. Control experiments in which small aliquots of ribosomal protein were radiolabeled and combined with a larger aliquot of the original sample before electrophoresis showed that the labeling reaction had no significant effect on the electrophoretie mobilities of the proteins in this electrophoretic system (Matthews et al. 1982).

For the purpose of this study, we have used the bovine proteins as a comparison base, identifying putative homologues in the rat on the basis of their electrophoretic mobilities. The relative mobilities of the radiolabeled rat and stained cow ribosomal proteins were obtained from the precise superposition of the fluorogram with the stained gel. These patterns were then ex- amined for protein pairs, which were scored as "identical" (cross- hatched spots, Fig. |) or "shifted" (large arrow, Fig, 1), in cases where rat proteins partially overlap or lie in the vicinity of a bovine protein. This approach is uncomplicated, provided such counterparts can be identified. However, in those instances where no radiolabeled or stained r-protein can be identified in the rat, such proteins are excluded from the pairwise analysis.

Results and Discussion

Comparison of Proteins in Cytoplasmic Ribosomes

T h e p r o t e i n s o f b o v i n e a n d r a t c y t o p l a s m i c r i b o -

s o m e s were c o m p a r e d b y c o e l e c t r o p h o r e s i s in t he s a m e two d i m e n s i o n a l e l e c t r o p h o r e s i s s y s t e m used to c o m p a r e the m i t o c h o n d r i a l r - p r o t e i n s f r o m these a n i m a l s ( E x p e r i m e n t a l P roc e du re s ) . S h o w n in Fig. I a re e l e c t r o p h e r o g r a m s o f p r o t e i n s f r o m the s m a l l a n d large s u b u n i t s o f b o v i n e c y t o p l a s m i c r i b o s o m e s , a l o n g w i t h the f l u o r o g r a m s o f t he se gels, d i s c lo s ing the corresponding r a d i o l a b e l e d ra t p ro t e in s . T h e ove ra l l d i s t r i b u t i o n a n d p a t t e r n o f i n d i v i d u a l p r o - t e ins in the s t a i n e d gel a n d f l u o r o g r a m is s imi l a r , i n d i c a t i n g t ha t c o r r e s p o n d i n g r - p r o t e i n s in these two

112

Fig. 1. Relative electrophoretic positions of proteins from rat and cow cytoplasmic ribosomes. Trace amounts of [~4C]-labeled rat r-proteins were mixed with bovine r-proteins before analysis of the mixtures by two dimensional polyacrylamide gel electrophoresis and fluorography (Experimental Procedures). The electrophoretic pattern of bovine proteins (open spots) from the small (A) and the large (D) subunits co-electrophoresed with a trace amount of[z4C]-labeled rat proteins from the small and the large subunits, respectively; B and E, fluorograms of the gels shown in A and D, respectively; C and F, schematic diagrams showing the relative electrophoretic positions of the bovine (open spots) and rat (solid spots) proteins from the small and large subunits, respectively. Overlapping spots are hatched; large and small arrows indicate bovine and rat proteins with major and minor mobility shifts, respectively. The locations of poorly visualized proteins (dashes) were verified in the reciprocal experiment using stained rat proteins co-electrophoresed with [~4C]-labeled bovine proteins (data not shown)

animals have similar electrophoret ic mobilities. In- deed, superposit ion o f the fluorograms (Fig. 1B and E) with the stained gels (Fig. 1A and D) reveals that most of the corresponding cow and rat proteins have identical mobilit ies in this system (Fig. 1C and F). However , some mobil i ty differences are discernible by this method. These differences range f rom relatively minor shifts (small arrows in Fig. 1 F) to major displacements in the electrophoretic pattern (large arrows). Using this approach o f pairwise compari - sons of bovine and rat proteins, we can identify two proteins in the small subunit o f rat cytoplasmic ribosomes that have altered mobilities. One o f these rat proteins fails to label under these condit ions (arrow, Fig. 1C), but this protein was found to have an altered mobili ty, based on the reciprocal experiment (data not shown) using radiolabeled cow proteins and stained rat proteins.

In a similar fashion, most o f the large subunit proteins o f the rat and the cow cytoplasmic ribosomes are comigratory (Fig. 1F). By pairwise comparison o f the bovine (Fig. 1D) and rat (Fig. 1E) proteins we can identify 8 proteins in the large subunit o f the rat cytoplasmic r ibosome that have altered mobilities. As shown in Fig. 1 F, three proteir~ pairs display relatively minor displacements (small arrows), while the remaining five protein pairs shove larger shifts (large arrows).

Using this co-electrophoretic technique to corn- pare the mobili t ies of cytoplasmic r ibosomal proteins f rom the cow and the rat, we find the majori ty of the proteins have identical mobilities, consistent with the idea that cytor ibosomal proteins are highly conserved (Delaunay et al. 1973; Fujisawa and Eli- ceiri 1975; Ramjou6 and Gordon 1977; Schiffmarl and Horak 1978; Madjar and Trau t 1980).

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FiR. 2. Relative electrophoretic posit ions o f proteins from rat and cow mitochondrial r ibosomes. Trace amounts of[~4C]-labeled rat r 'pr~ were mixed with bovine r-proteins and the mixtures were analyzed by two dimensional polyacrylamide gel electrophoresis ~ d fluorography (Experimental Procedures). Electrophoretic pat terns o f bovine proteins from the small (A) and the large (D) subunits COeleet ~4 eros from the small and the large subunits, respectively B and E tt- rophoresed with a trace amount o f [ C]-labeled rat prot " . , . , ; ,

uoro m~ a e t h , ,o1~ ~,^, ~ ;~ A on~ r~ r~n~e ' v F schematic dia ares showing the relative e~ectropnoretic posit ions _ e r a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . t ivel . ; C and , gr ~r the bovine (open spots) and rat (solid spots) proteins from the small and the large subunits, respectively. Overlapping spots are uatChed; comigratory proteins are identified using the bovine mitor ibosomal n u m b e n n g system (Matthews et al. 1982)

Comparison of Proteins in Mitochondrial Ribosomes

The proteins of bovine and rat mitochondrial ri- bt~SOmes were also compared by the same technique, ~sing the well-characterized bovine mitochondrial ~bosome (Matthews et al. 1982) as a comparison

ase for the pairwise analysis of proteins. Shown in l~ig, 2 are the electropherograms of proteins from the Small and large subunits of the bovine mito- Chondrial ribosome, along with the fluorograms of these gels disclosing the corresponding radiolabeled rat mitoribosomal proteins. While the overall electrophoretic pattern of proteins in the stained gels and ltUorograms is similar, superposition of the fluorograms (Fig. 2B and E) with the stained gels (Fig. 2A and D) reveals that most of the cow and rat ribosomal proteins have very different mobilities.

Using the procedure of pairwise comparison, we can identify only four proteins in the small subunit

of the rat mitochondrial ribosome that have the same mobilities as a corresponding bovine protein (Fig. 2C). These bovine proteins are S13, S17, $26, and $32 (Matthews et al. 1982). Following the same approach, most of the proteins in the large subunits of the rat and the cow mitochondrial ribosomes ap- pear to have different mobilities (Fig. 2F). The pairwise comparison of the radiolabeled rat proteins (Fig. 2E) with the stained bovine proteins (Fig. 2D) reveals only five rat proteins comigrating with bovine proteins. These bovine proteins are L3, L6, L22, L41, and L51/52 (Fig. 2F).

Rate of Evolution of Mitochondrial and Cytoplasmic Ribosomal Proteins

The results of the pairwise analysis of individual r-proteins in the cow and the rat are summarized in Table 1. While most of the proteins in cytoplas-

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Table 1. Predicted rates of change of proteins in mitochondrial and cytoplasmic ribosomes of the cow and the rat

Ribosome

No. of proteins in Expected no. of mobility class Fraction detectable dif-

conserved ferences per Subunit Comigratory Altered I protvin D

Cytoplasmic Small 22 2 0.92 0.08 +_ 0.06 Large 44 8 0.84 0.16 + 0.06 Both 66 10 0.87 0.14 + 0.04

Mitochondrial Small 4 25 0.14 1.97 _+ 0.46 Large 6 31 0.16 1.83 _+ 0.38 Both 10 56 0.15 1.88 __ 0.29

D, the mean number of detectable electrophoretic differences expected per protein, is calculated from the fraction of identical proteins (/) using the relationship D = -In L The standard error for D is given by [(1 - I)/nl] v', where n is the number of proteins compared (Nei 1971)

mic ribosomes of these two species have identical mobilities, only 15% of the mitoribosomal proteins have conserved electrophoretic properties. These findings indicate that the mitoribosomal proteins have acquired more amino acid changes than cytoplasmic r-proteins. Thus, a reasonable prediction that can be made on the basis of these results is that proteins of the mitochondriaI ribosome have accumulated extensive changes in the primary amino acid sequence since the divergence of the cow and the rat about 73 million years ago (McLaughlin and Dayhoff 1972). In addition, it is evident that the proteins ofmitochondrial ribosomes are, as a group, diverging more rapidly than those in cytoplasmic ribosomes.

In the absence of amino acicl sequence infor- mation, the electrophoretic properties of homologous proteins in two species can be used to evaluate the relative rate of change (Nei 1971). Using this approach, the rate of divergence of mitoribosomal proteins relative to cytoribosomal proteins can be assessed. For this comparison, we used the relationship

D = - l n I (1)

(Nei 1971) where D is the mean predicted number of electrophoretically detectable differences per protein, and I is the observed fraction of electrophoretically identical proteins. However, the D values predicted are expected to underestimate the actual number of amino acid substitutions in the individual proteins for two reasons: (1) not all amino acid substitutions will result in electrophoretically detectable differences, and (2) all mobility shifts are scored equally in this analysis, irrespective of the number of amino acid substitutions in each protein. Nevertheless, the D values should be proportional to the number of amino acid changes accumulated in the proteins from these two species. Given these limitations, we can, however, estimate the relative rates of change of r-proteins in mitochondrial and cytoplasmic ribosomes by comparing the D values.

As shown in Table 1, the D value calculated for the cytoplasmic ribosome is low (0.14) compared to that estimated for the mitochondrial ribosome (1.88). From the ratio Dmj~/Dcyt, it is apparent that mitoribosomaI proteins are accumulating changes at a rate about 13 times higher than that of cytoplasmic ribosomes.

This difference in rates could reflect an intrinsi- cally high evolutionary rate of mitoribosomal proteins, a relatively low rate for cytoribosomal proteins, or a combination of both factors. Undoubtedly, the conserved nature of cytoribosomal proteins, in- ferred from earlier studies (Delaunay et al. 1973; Fujisawa and Eliceiri 1975; Ramjou6 and Gordon 1977; Schiffman and Horak 1978; Madjar and Traut 1980), contributes significantly to the observed difference in evolutionary rates for mitochondrial and cytoplasmic r-proteins.

In Table 2, we compare the results of the electrophoretic analysis of rat and cow cytoplasmic r-proteins with similar electrophoretic analyses in different vertebrates. The mean number of predicted differences per protein (D) calculated from rat/coW comparison [0.141] is in the same range as values calculated from rat/rabbit [0.093] (Madjar and Traut 1980) and rat/chicken comparisons [0.33] (Ramj ou~ and Gordon 1977). The higher value for the rat/ chicken comparison reflects the longer time of divergence of these species. When corrected for divergence times, the results of these three indepen- dent studies agree closely (Table 2), indicating that the cytoplasmic r-proteins in these species are evolving at similar rates.

The actual rate of amino acid substitution for cytoplasmic r-proteins can be determined by comparing the sequences of homologous r-proteins. The amino acid sequence is known for the homologueS of the E. coli r-protein L7/12 in yeast (YPAI) (Itoh 1980), Artemia (eL12) (Amons et al. 1974) and the rat (P2) (Linet al. 1982). The rat andAr temia homologues differ in 43 of their 111 residues, while the rat and yeast proteins differ at 58 sites (Table 3).

Table 2. Predicted rates of change of proteins in cytoplasmic ribosomes of vertebrates a

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No. proteins in Fraction mobil iw class conserved

Comparison b Comigratory Altered I D - . - . . . ._

Expected no. detectable differences per protein

Normalized D x 10-9/ year

Rat/cow 66 10 0.868 0.141 0.97 Rat/rabbit 72 7 0.911 0.093 0.71 Rat/chicken 61 24 0.718 0.33 0.55

The mean number &detectable electrophoretic differences, D, is calculated from the fraction of identical proteins, I, as in Table 1. The D values in each comparison were normalized using the relationship, D.o~ = D/2T, where T, the divergence time for each

b eOraParison is' rat /rabbit = 65 million years rat/cow = 73 million years, and rat/chicken = 300 million years (Dayhoff 1978) he source of data for each comparison is: rat/cow (this study); rat/rabbit (Madjar and Traut 1980); rat/chicken (Ramjoue and

Gordon 1977)

Table 3. Observed rates of change of proteins in cytoplasmic ribosomes

Rate of amino acid substitution per

No. different b amino Fraction altered Fraction identical site per year Comparison. acid residues D I k,~

Rat P2/ drternia eLl2 43 0.387 0.616 3.5 x 10 -~o

Rat P2/ RYeast YPA1 58 0.523 0.477 3.1 x 10 -z0

at L37/ Yeast YP55 40 0.459 0.540 2.6 x 10-,o

' The divergence time used for the rat and Artemia salina is 700 million years (Dayhoff 1978), and 1200 million years for the rat and Yeast (Osawa and Hori 1980)

b Total number of residues in proteins compared is 111 for the P2 homologues (Lin et at. 1982) and 87 for L37 (Lin et at. 1983)

The amino acid sequence is also known for the rat ribosomal protein L37 and its yeast homologue u which differ in 40 of their 87 residues (Lin et al. 1983). These values can be used to calculate the rate of amino acid subsitution, kaa, using the equatio n

kaa = -ln(1 - d~)/2T (2)

Where do is the fraction of altered residues in the l~roteins compared and T is the time of divergence ~ two species (Zuckerkandl and Pauling 1965). C~rnpared to the rat protein P2, the Artemia and Yeast homologues have similar rates of amino acid SUbstitution (Table 3), averaging 3.3 • 10 -~~ substitutions per site per year. The fact that this rate is the Same as that calculated for the B. subtilis ho- ra~ of E. coli L7/12 (Osawa and Hori 1980) SUggests that similar functional constraints govern ~ e evolution of this protein in diverse organisms.

addition, the yeast homologue of the rat protein 37 is ble 3 This low rate �9 similarly conserved (Ta ). "

of amino acid substitution places these ribosomal l~roteins in the class of highly conserved proteins (Wilson et al. 1977), along with cytochrome c (k~ = 2.2 • 10-~0 substitutions per site per year).

On the basis of the electrophoretic comparisons in this study, we determined that mitochondrial r-proteins are changing at a rate 13 times that of the cytoplasmic r-proteins. This rate corresponds to an amino acid substitution rate of 40 x 10 -~0, using the average ka~ value of 3.06 x 10 -1o calculated above as a representative value for cytoplasmic r-proteins. This evolutionary rate for mitoriboso- real proteins is indeed high, when compared with amino acid substitution rates for other proteins, such as myoglobin (9 • 10-1~ albumin (19 x 10-~0), and pancreatic RNase (21 • 10 -t~ (Wilson et al. 1977). In fact, the evolutionary rate for mitoribosomal proteins places them in the category of rapidly evolving proteins, exceeded only by rates for neu- rotoxins, fibrinopeptides, and immunoglobul in variable chains (Wilson et al. 1977).

Ribosomal RNA and Protein Evolutionary Rates

To determine evolutionary rates for the RNA in mammalian mitoribosomes, we compared the sequences available for the rat (Kobayashi et al. 1981; Saccone et at. 1981), cow (Anderson et al. 1982), and human (Anderson et al. 1981) mitoribosomal

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Table 4. Evolutionary rates of mitochondrial and cytoplasmic ribosomal RNA*

Comparison rRNA

Substitu- tions per

Fraction site per different year nucleo- Nucleo-

Percent tides tide homology d, k, x 10 -~o

Mitochondrial Rat/cow 12S 78.6 0.214 17

16S 75.6 0.244 20 Rat/human 12S 76.5 0.235 19

16S 73.0 0.270 23 Human/cow 12S 78.5 0.215 17

16S 78.0 0.220 18 Cytoplasmic

Rat/human 18S 98.8 0.012 0.8

* The divergence time for the rat, cow and human lineages is considered to be about 73 million years (Dayhoff 1978) Nueleotide sequences used: rat 12S (Kobayashi et al. 1981) and 16 S (Saceone et al. 1981); cow 12S and 16S (Anderson et al. 1982); human 12S and 16S (Anderson et al. 1981); rat 18S (Chan et al. 1984) and human 18S (Torezynski et al. 1985)

kn = -3/4 In(l - 4/3 d,) where d, is the fraction of different 2T

residues (Jukes and Cantor 1969). Nucleotide sequence homologies were determined using the SEQA program from the IDEAS DATABASE, Los Alamos Sequence Analysis Package for Nucleic Acids and Proteins (Ka- nehisa 1982), except for the value reported for the rat/human 18S comparison (Torczynski et al. 1985)

R N A s (Table 4). In addit ion, the sequences that are available for 18S R N A in h u m a n (Torczynski et al. 1985), and rat (Chan et al. 1984) cy toplasmic ribosomes also allows es t imat ion o f the evolut ionary rate for m a m m a l i a n cy toplasmic rRNAs. The nucleotide subst i tut ion rates, kn, for these r R N A s were calculated (Table 4) using the relat ionship:

- 3/4 ln(l - 4/3 dn) kn = (3)

2 T

where dn is the fraction of different nucleotides and T is the divergence t ime for the species c o m p a r e d (Jukes and Cantor 1969). The rates o f the nucleotide substi tut ion for these mi to r ibosoma l R N A s range f rom 17 x 10 - m to 23 x 10 -1o subst i tut ions per site per year, indicating that they are evolving at rates about 23 t imes that o f the more conserved cytoplasmic r R N A s (Table 4).

The result that the mi to r ibosoma l R N A and proteins are changing at comparab le rates is especially interesting in view o f the fact that they are products o f different genomes, where muta t iona l rates are es t imated to differ three to ten fold (Brown et al. 1979; Miyata et al. 1982; Lanave et al. 1985). C o m - par ison o f the r -protein and r ibosomal R N A rates

of evolu t ion suggest that changes are being fixed at comparab le rates in bo th the R N A and prote in components o fmi tochondr i a l r ibosomes , despite the different muta t iona l rates for the R N A and protein. This implies that functional constraints act more or less equally on bo th kinds of molecules in the ribosome. A s imilar relat ion also holds for cytoplasmic r ibosomes in which, despite their overal l higher degree o f conservat ion, the R N A s (Table 4) and proteins (Table 3) show essentially the same evolut ionary rate. Such concordan t evolu t ion o f R N A and prote in c o m p o n e n t s in two kinds o f r ibosomes, which are evolving at different rates, implies that the proteins, as well as the RNA, are subjected to effectively s imilar s t ructural / funct ional constraints.

I t is evident that r ibosomes of m a m m a l i a n mi- tochondr ia are evolving m o r e rapidly than their ex- t rami tochondr ia l counterpar ts in the same cell. This is true for the protein componen ts , found in this s tudy to be changing at a 13 fold higher rate than that for the cy toplasmic r-proteins as well as for the rRNA. This finding is especially interesting, since both sets o f r i b o s o m a I proteins, mi tochondr ia l and cytoplasmic, are encoded by nuclear genes (Schieber and O'Br ien 1985). The difference in evolutionarY rates implies that they are encoded by different setS of genes and that functional constraints operating on r ibosomes are relaxed for mi tochondr ia l ribosomes. This p h e n o m e n o n reflects the nature of the t ranslat ion products o f ei ther r ibosome, and can be explained by the absence of a potent ial error cascade in mi tochondr ia (Hasegawa et al. 1984; Wilson et al. 1985). I t is certainly less critical for the mitochondrial r i bosome to main ta in a high degree of t ranslat ional accuracy, since no molecules required for mi tochondr ia l gene expression or D N A repli- cation are m a d e on mi tochondr ia l r ibosomes. Rath- er, these r ibosomes synthesize mult iple copies of only a l imited set (thirteen) of prote in componen t s of the energy t ransduct ion system (Chomyn et al. 1985). With such a r edundancy o f nonregulatorY molecules, occasional mis takes in mi tochondr ia l prote in synthesis are not likely to have deleterious consequences for the cell.

Acknowledgments. The authors thank Karen Dukes for her help in preparing the manuscript. This research was supported bY USPHS NIH Grants GM-15438 and GM-23322.

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evolution of proteins in mammalian cytoplasmic and mitochondrial ribosomes

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