Biochem. Physiol. Pflanzen 175, 1-8 (1980)
Analysis of Ribosomal Proteins from Various Species of Algae
Comparative Electrophoretic Study
on Proteins from Chloroplast Ribosomes
HELMUT GOTZ and CARL-GEROLD ARNOLD
Institut fiir Botanik und Pharmazeutische Biologie der Universitat Erlangen-Niirnberg,
Federal Republic of Germany
Key Term Index: chloroplast ribosomal subunits, evolution rates, ribosomal proteins, two-dimensional gel electrophoresis; unicellular algae.
Summary
The proteins from chloroplast ribosomal subunits of seven species of algae were characterized
by two-dimensional gel electrophoresis. The protein patterns from the chloroplast ribosomal subunits
were compared to those of Chlamydomonas reinhardii. A high degree of evolutionary conservation
was found among the ribosomal proteins of the different species. For Chlamydomonas eugametos 26,
Chlamydomonas noctigama 27, Chlorogonium elongatum 30, Scenedesmus obliquus 25, Euglena gracilis
18 proteins were found to be homologous to corresponding proteins in Chlamydomonas reinhardii.
On the whole, the chloroplast ribosomal protein patterns differed slightly more on average than the
cytoplasmic ribosomal protein patterns of these species. No homologies were detected between
chloroplast and cytoplasmic ribosomal proteins of the same species.
Introduction
A characteristic feature of eukaryotes is the existence of organelles, such os chloroplasts and mitochondria in the cytoplasm. Choroplosts have been found to demonstrate
striking structural and functional similarities to prokaryotes in photosynthetic membranes (ARNTZEN and BRIANTIS 1975), in pathways of CO 2 fixation and photo~ynthetic
electron flow (TAYLOR 1970) and in ribosomes (LOENING and INGLE 1967; SPIESS and
ARNOLD 1975).
Chloroplast ribosomes were first isolated from spinach (LYTTLETON 1962); since
then they have been isolated and characterized in several higher plants (BOULTER et al.
1972; GUALERZI et al. 1974). In contrast to 80S ribosomes in the cytoplasm, both chloroplast ribosomes and ribosomes of prokaryotes have sedimentation coefficients of approximately 70S. Furthermore, they are sensitive to antibiotics which inhibit protein
synthesis on bacterial ribosomes but are resistant to those which interfere with 80S
ribosomal functions. Because of these similarities to prokaryotes, chloroplast ribosomes
are often referred to as "prokaryotic ribosomes". Thus it would be of interest to see to
what extent evolutionary changes take place in the chloroplast ribosomes, which are
possibly less exposed to the factors of selection during evolution than the cytoplasmic
ribosomes.
In our study we investigated the ribosomal proteins of chloroplast ribosomes in seven
species of algae: the Volvocales Chlamydomonas reinhardii, Chlamydomonas eugametos,
1 Bioehem. Physio!. Pflanzen, Bd. 175
2
H. GiiTZ and C.-G. ARNOLD
Chlamydomonas noctigama, Chlorogonium elongatum, the Chlorococcales Scenedesrnus
obliquus, Chlorella fusca and Euglena gracilis which is not ranged among the Chlorophyceae class. We compared the protein patterns obtained by two-dimensional gel
electrophoresis both with each other and with their cytoplasmic ribosome counterparts.
The results of the electrophoretic study of algal cytoplasmic ribosomes were reported in
an earlier paper (GOTZ and ARNOLD 1980).
Material and Methods
The strains and conditions for cultivating algae were the same as described by GiiTZ and ARNOLD
(1980). The separation of the ribosomal subunits from Chiarella fusca and Euglena gracilis was done
essentially according to HANSON et al. (1974), but a small zonal rotor (Spinco Ti-14) was used. About
200 A260 units of ribosomes were layered over an exponential sucrose gradient of 5-30% (wjv)
sucrose and centrifuged at 172,000· g for 4 h. The small subunit of chloroplast ribosomes from Chiarella fusca proved to be very unstable and degraded, whereas the large subunit could be isolated in
sufficient amounts. Separation of chloroplast ribosomes from cytoplasmic ribosomes in the other
organisms was achieved by sucrose density centrifugation in a gradient of 5-30% (w/v) sucrose.
Ribosomal subunits were isolated from an exponential sucrose gradient in the Spinco Ti-14 zonal
rotor by a dense sucrose solution passing through a flow-cell. The mitochondrial ribosomes dissociate
under the same conditions as the chloroplast ribosomes, and since mitochondrial ribosomes make up
only 1 % of the total number of ribosomes (BOURQUE et al. 1971), our 70S ribosomal preparations are
considered to contain predominantly chloroplast ribosomes. The analyses of purity of ribosomal
subunits, extraction of ribosomal proteins and their characterization by two-dimensional gel electrophoresis were as reported earlier (GiiTZ and ARNOLD 1980).
Results and Discussion
As shown in Table 1, the number of ribosomal proteins in the large and the small
subunits lie in a very narrow range for all the analysed species. Although we found
sedimentation values of 528 for the large and 378 for the small ribosomal subunits,
with the exception of Euglena gracilis, whose small subunit sedimented at 32S, we
designated the subunits uniformly as 50S and 30S respectively. The small deviations
Table 1. Number of proteins in the large and the small chloroplast ribosomal subunits of various species
of algae
Species
Number of proteins
50S
30S
References
Chlamydomonas reinhardii
26
34
30
26
23
30
26
26
28
33
30-34
HANSON et al. (1974)
BRUGGER and ROSCHETTI (1975)
SPIESS (1977)
this paper
this paper
this paper
this paper
this paper
this paper
this paper
FREYSSINET (1977)
Chlamydomonas eugametos
Chlamydomonas noctigama
Chlorogonium elollgatum
Scenedesmus obliquus
Chlorella fusca
Euglena gracil'is
22
25
20
22
23
21
24
27
25
22-24
Analysis of Algal Ribosome Proteins
3
in the number of proteins for ribosomal subunits given by various authors could be due to
the different conditions of protein extraction and electrophoresis (GOTZ and ARNOLD 1980).
Figures 1 and 2 show the schematic protein patterns obtained by two-dimensional
electrophoresis from the large and the small ribosomal subunits. The location of the
spots on the gel plates in the second dimension reveal that the chloroplast ribosomal
proteins of the analysed algae have molecular weights between 10,000 and 52,000 as
compared to proteins of known molecular weights.
The protein patterns of both ribosomal subunits suggest a high degree of evolutionary
conservation. The evolution rates of the chloroplast ribosomal proteins range from
0.40 to 0.60 compared to the reference of Chlamydomonas reinhardii which was given
a value of 1.0. If we arrange the analysed organisms according to the number of chloroplast ribosomal proteins which are homologous to each other, we find the following
decreasing degree of affinity: Chlamydomonas reinhardii - Chlorogonium elongatum Chlamydomonas noctigama - Chlamydomonas eugametos - Scenedesmus obliquus Chlorella fusca - Euglena gracilis.
The order of arrangement coincides roughly with the taxonomic classification of
these algae in the plant system, it is only remarkable how similar Chlorogonium elongatum and Chlamydomonas reinhardii are to each other although they belong to different
genera. Nevertheless, the ribosomal proteins can not be used as the sole criterion for the
classification of different species, because the differences of proteins on the gel plates
are not strictly proportional to the taxonomic differences of the species. There are also
more uncertainties involved, in that, the deviations on the gel plates only indicate
that two proteins are different, but do not quantify the differences themselves, e.g.
do not quantify the actual number of different amino acids. Furthermore, it is sometimes difficult to distinguish between one spot and two closely overlapping spots, since
ribosomal proteins are not present in equal stoichiometric amounts (DEUSSER et al.
1974; KRUISWIJK et al. 1978).
It is a well-known fact that chloroplasts and mitochondria synthesize proteins
(TEWARI 1971; HARTLEY et al. 1975; LAMBOWITZ et al. 1976). The protein synthesizing
processes and the protein synthesizing machinery of these organelles resemble those
of bacteria more closely than those of the cytoplasm of eukaryotic organisms. Protein
synthesis by chloroplasts, mitochondria, and bacteria is carried out on ribosomes of the
70S type, while protein synthesis of the cytoplasm of eukaryotes is carried out on
ribosomes of the 80S type. The question of whether there are any similarities between
proteins of organelle ribosomes and those of cytoplasmic ribosomes is important from
the aspect of the biogenesis of these ribosomes. It is conceivable that there may be a
few common or structurally related proteins in the two ribosomal populations in the
same cell, and some of the proteins (eventually formed in cytoplasmic ribosomes) may
have been processed further, either by limited peptide cleavage or by secondary modification, upon their entry into the organelle. Only a few works treat this question and the
available results are still inconclusive.
GUALERZI et al. (1974) found little resemblance between the protein patterns of
chloroplast and cytoplasmic ribosomes of bean, wheat and spinach. Similar results
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Analysis of Algal Ribosome Proteins
5
have also been reported in other higher plants (VASCONCELOS and BOGORAD 1971).
In contrast, JANDA and WITTMANN (1968) found good agreement between protein profiles of chloroplast and cytoplasmic ribosomes isolated from spinach, while this agreement was less striking in the case of beans.
For mitochondrial ribosomes, fractionation of the proteins by gel electrophoresis
showed that they are significantly different from those of the cytoplasmic ribosomes
(LIZARDI and LUCK 1972; ISJIIGURO and ARAKATSU 1975) and that the degree of evolutionary divergence among mitochondrial ribosomal proteins is much higher than among
the cytoplasmic ribosomal proteins in the same organisms (MATTHEWS et al. 1978).
In our experiments we found little homology between chloroplast and cytoplasmic
(GOTZ and ARNOLD 1980) ribosomal proteins in the same species, whereas a high degree
of similarity could be detected for the proteins of single subunits throughout all the
species investigated. Thereby, the conformity of the protein patterns for chloroplast
ribosomal subunits as well as for cytoplasmic ribosomal subunits is roughly in accordance
with the taxonomic position of the species.
Although the 70S ribosomes of prokaryotes and chloroplasts of eukaryotes seem
to be homolog'ous (GRAY and HERSON 1976; LEE and EVANS 1971; GRIVELL and WALG
1972), it is remarkable that no homologies exist between the 80S ribosomes in the cytoplasm of eukaryotes and the 70S ribosomes in the chloroplasts of eukaryotes. This difference can be explained with the en do symbiotic hypothesis, which states that chloroplasts
arise during evolution through symbiosis between a prokaryote and a primitive eukaryotic cell. Therefore, according to this hypothesis, ribosomes from the organelles and the
cytoplasm would be ribosomes from originally separate organisms.
The endosymbiote hypothesis cannot be directly proved, but might be more probable
if different evolution rates of the symbiosis partners in the same organisms could be
determined. Our results do not confirm this conjecture, because the protein patterns
of the plastid ribosomes and the cytoplasm ribosomes differ in nearly the same proportions from one organism to another; however, these results also do not repudiate the
endosymbiote hypothesis, because it has been proved that the synthesis of the majority
of ribosomal proteins from chloroplasts are coded by the nucleus and not by the genome
of the organelles (APEL and SCHWEIGER 1972; KLOPPSTECH and SCHWEIGER 1973;
BOURQUE and WILDMAN 1973; HONEYCUTT and MARGULIES 1973; FREYSSINET 1978).
This nuclear coding then could explain the similar evolutionary process of chloroplast
and cytoplasmic ribosomal proteins within the realm of the endosymbiote hypothesis.
Fig. la-g. Analysis of proteins from chloroplast 508 ribosomal subunits.
The diagrams show the schematic protein patterns obtained by two-dimensional electrophoresis
from Chlamydomonas reinhardii (a), Chlamydomonas eugametos (b), Chlamydomonas nociigama (c),
Chlorogonium elongatum (d), 8cenedesmus obll:quus (e), Chlorella fusca (f), Euglena gracilis (g). The
filled-in circles represent the proteins which are identical with proteins in Chlamydomonas reinhardii
on the gel plates and the cross-hatched circles represent the proteins which are similar and correlate
with equivalent proteins in Chlamydomonas reinhardii.
6
H. GOTZ and C.-G.
ARNOLD
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Fig. 2a-f. Analysis of proteins from chloroplast 308 ribosomal subunits.
The diagrams show the schematic protein patterns obtained by two-dimensional electrophoresis from
Chlamydomonas reinhardii (a), Chlamydomonas eugametos (b), Chlamydomonas noctigama (c), Chlorogonium elol1gatum (d), 8cenedesmus obliquus (e), Euglena gracilis (f). The filled-in circles represent the
proteins, which are identical with proteins in Chlamydomonas reinhardii on the gel plates and the
cross-hatched circles represent the proteins which are similar and correlate with equivalent proteins
in Chlamydomonas reinhardii.
Analysis of Algal Ribosome Proteins
7
If we inspect the evolution rates of characteristics which are coded by the organelle
genome, the en do symbiote hypothesis becomes much clearer. TAKABE and AKAZAWA
(1975) and BROWN et al. (1976) investigated the structural homology of the two subunits
of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase from various origins.
It was found that the large subunit of the enzyme, which is coded by the chloroplast
genome, is structurally homologous during phylogenetic evolution, whereas the small
subunit, which is coded by the nucleus, varies considerably in the different species.
These results show that the evolution rates of the chloroplast genome progress more
slowly than the rates of the nuclear genome. This would agree with the results of PIGOTT
and CARR (1972), who have found many homologies between the primary structures of
the chloroplast coded rRNA of Euglena chloroplasts and the rRNAs in various blue-green algae. On the other hand,jt is surprising that antisera of spinach chloroplast
ribosomal proteins show no immunological cross-reaction with the ribosomal proteins
of bacteria and blue-green algae (GuALERzI et al. 1974). If there were no differences in
the sensitivity of the techniques used, these results could possibly indicate that, during
evolution, the rRNA is more extensively conserved than the ribosomal proteins.
With this in mind, we feel our investigations should be continued with the RNA
portion of ribosomes in order to make clear the course of evolution of the chloroplast
and cytoplasmic rRNAs in these species of algae.
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8
H. GOTZ and C.-G. ARNOLD, Analysis of Algal Ribosome Proteins
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Received July 28, 1979.
Author's address: Dr. HELMUT GOTZ and Prof. Dr. CARL-GEROLD ARNOLD, Institut fiir Botanik und
Pharmazeutische Biologie der Universitiit Erlangen-Niirnberg, SchloBgarten 4, D - 8520 Erlangen.