Partial characterization of the biosynthesis and integration of the Photosystem II reaction centers in the thylakoid membrane of Chlamydomonas reinhardtii

Pulse-labeling of wild-type and a Photosystem II mutant strain of Chlamydomonas reinhardtii was carried out in the presence or absence of inhibitors of either cytoplasmic or chloroplast ribosomes, and their thylakoid membrane polypeptides were analyzed by polyacrylamide gel electrophoresis. A pulse-chase study was also done on the wild-type strain in the presence of anisomycin, an inhibitor of protein synthesis on cytoplasmic ribosomes. The following results were obtained: the Photosystem II reaction center is mainly composed of integral membrane proteins synthesized within the chloroplast. Several of the proteins of the Photosystem II reaction center are post-translationally modified, after they have been inserted in the thylakoid membrane.     

Properties and topography of the major integral plasma membrane protein of a unicellular organism

The cellular distribution, membrane orientation, and biochemical properties of the two major NaOH-insoluble (integral) plasma membrane proteins of Euglena are detailed. We present evidence which suggests that these two polypeptides (Mr 68 and 39 kD) are dimer and monomer of the same protein: (a) Antibodies directed against either the 68- or the 39-kD polypeptide bind to both 68- and 39-kD bands in Western blots. (b) Trypsin digests of the 68- and 39-kD polypeptides yield similar peptide fragments. (c) The 68- and 39-kD polypeptides interconvert during successive electrophoresis runs in the presence of SDS and beta- mercaptoethanol. (d) The 39-kD band is the only major integral membrane protein evident after isoelectric focusing in acrylamide gels. The apparent shift from 68 to 39 kD in focusing gels has been duplicated in denaturing SDS gels by adding ampholyte solutions directly to the protein samples. The membrane orientation of the 39-kD protein and its 68-kD dimer has been assessed by radioiodination in situ using intact cells or purified plasma membranes. Putative monomers and dimers are labeled only when the cytoplasmic side of the membrane is exposed. These results together with trypsin digestion data suggest that the 39- kD protein and its dimer have an asymmetric membrane orientation with a substantial cytoplasmic domain but with no detectable extracellular region. Immunolabeling of sectioned cells indicates that the plasma membrane is the only cellular membrane with significant amounts of 39- kD protein. No major 68- or 39-kD polypeptide bands are evident in SDS acrylamide gels or immunoblots of electrophoresed whole flagella or preparations enriched in flagellar membrane vesicles, nor is there a detectable shift in any flagellar polypeptide in the presence of ampholyte solutions. These findings are considered with respect to the well-known internal crystalline organization of the euglenoid plasma membrane and to the potential for these proteins to serve as anchors for membrane skeletal proteins.     

Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.     

Influence of photosynthesis and chlorophyll synthesis on polypeptide accumulation in greening euglena

Two-dimensional gel electrophoresis resolves total cellular protein from Euglena gracilis klebs var bacillaris Cori into 640 polypeptides, 79 of which are induced by light exposure. The inhibition of chloroplast translation by streptomycin, the direct inhibition of photosynthesis as well as the indirect inhibition of chlorophyll synthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the specific inhibition of photosynthesis but not chlorophyll synthesis by DCMU in the presence of 17 millimolar ethanol failed to inhibit the accumulation of 40 polypeptides. These polypeptides appear to be synthesized on cytoplasmic ribosomes and their accumulation is independent of the developmental status of the chloroplast. Streptomycin but not DCMU completely inhibited the accumulation of six polypeptides which are undetectable in mutants lacking chloroplast DNA suggesting that these polypeptides are translated on chloroplast ribosomes. The accumulation of seven polypeptides which are detectable in mutants lacking chloroplast DNA was also inhibited by streptomycin but not by DCMU suggesting that the accumulation of these polypeptides is dependent upon stabilization by a chloroplast translation product. The accumulation of 12 polypeptides was inhibited by streptomycin and by DCMU under conditions in which chlorophyll synthesis was inhibited, but not under conditions in which chlorophyll synthesis was unaffected by DCMU. The inhibition by DCMU of the accumulation of these polypeptides appears to be due to the inhibition of chlorophyll synthesis suggesting that they are components of pigment protein complexes. The accumulation of six polypeptides was inhibited under all conditions in which photosynthesis was inhibited suggesting that the accumulation of these polypeptides is dependent upon a product of photosynthesis.     

Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells.

Techniques are described for the isolation of plastid thylakoid membranes from light-grown and dark-grown cells of Euglena gracilis var. bacillaris, and from mutants affecting plastid development. These membranes, which have minimal contamination with other cell fractions, are localized in sucrose gradients by using the thylakoid membrane sulfolipid as a specific marker. The plastid thylakoid membrane polypeptides isolated from these membranes were separated on SDS polyacrylamide gels and yielded patterns containing 30-40 polypeptides. Light-grown strain Z gave patterns identical with bacillaris. Since the plastid thylakoid polypeptide patterns obtained from dark-grown wild-type cells and from a bleached mutant W3BUL in which plastid DNA is undetectable are identical, it appears that the proplastid thylakoid polypeptides of wild-type cannot be coded in plastid DNA and are probably coded in nuclear DNA. The plastid thylakoid polypeptide patterns obtained from various dark-grown mutants, making large but abnormal chloroplasts, show a correlation between the amount of chlorophyll formed and the amount of a plastid thylakoid polypeptide thought to be associated wtth one of the pigment-protein light-harvesting complexes. Treatment with SAN 9789 (4-chloro-5-(methylamino)-2(alpha, alpha, alpha,-trifluoro-m-tolyl)-3-(2H(pyridazinone) known to block carotenoid synthesis at the level of phytoene, causes a progressive loss of all plastid thylakoid polypeptides during growth in darkness and results in the establishment of a new, lowere steady-state level of sulfolipid. At least ten of the plastid thylakoid polypeptides become labeled when isolated chloroplasts are supplied with radioactive amono acids; of these six are undectable in W3BUL and are, therefore, candidates for coding by plastid DNA.     

盐胁迫下苜蓿中盐蛋白的诱导产生

盐胁迫下苜蓿叶片中蛋白质的合成受到抑制,而其离体叶绿体中蛋白质合成增强,ABA阻碍了后者的蛋白质合成。NaCl胁迫下,“松江”和“肇东”两品种的根和叶中均无新多肽出现。在盐敏感的“松江”品种离体叶绿体中,NaGl诱导70,65,60和43kD4种多肽产生,ABA诱导60和17kD两种多肽产生;在较抗盐的“肇东”品种离体叶绿体中,NaGl诱导83,80kD和43kD3种多肽产生,但100mmol/L NaCl并不诱导83kD多肽出现,ABA无明显作用。两品种的43kD多肽和肇东品种的80kD多肽都存在于类囊体膜上,而松江品种的60kD多肽则存在于叶绿体间质中。     

Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii

The ratio of free to thylakoid-bound chloroplast ribosomes in Chlamydomonas reinhardtii undergoes periodic changes during the synchronous light-dark cycle. In the light, when there is an increase in the chlorophyll content and synthesis of thylakoid membrane proteins, about 20-30% of the chloroplast ribosomes are bound to the thylakoid membranes. On the other hand, only a few or no bound ribosomes are present in the dark when there is no increase in the chlorophyll content. The ribosome-membrane interaction depends not only on the developmental stage of the cell but also on light. Thus, bound ribosomes were converted to the free variety after cultures at 4 h in the light had been transferred to the dark for 10 min. Conversely, a larger number of chloroplast ribosomes became attached to the membranes after cultures at 4 h in the dark had been illuminated for 10 min. Under normal conditions, when there was slow cooling of the cultures during cell harvesting, chloroplast polysomal runoff occurred in vivo leading to low levels of thylakoid-bound ribosomes. This polysomal runoff could be arrested by either rapid cooling of the cells or the addition of chloramphenicol or erythromycin. Each of these treatments prevented polypeptide chain elongation on chloroplast ribosomes and thus allowed the polyosomes to remain bound to the thylakoids. Addition of lincomycin, an inhibitor of chain initiation on 70S ribosomes, inhibited the assembly of polysome-thylakoid membrane complex in the light. These results support a model in which initiation of mRNA translation begins in the chloroplast stroma, and the polysome subsequently becomes attached to the thylakoid membrane. Upon natural chain termination, the chloroplast ribosomes are released from the membrane into the stroma.     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : III. Chloroplast Ribosomes and Membrane Organization

The fine structure of the ac-20 strain of Chlamydomonas reinhardi is described. Cells grown mixotrophically in the presence of acetate have a highly disordered chloroplast membrane organization and usually lack pyrenoids. Chloroplast ribosome levels are only 5–10% of wild-type levels. Cells grown phototrophically without acetate possess more chloroplast ribosomes and have more normal membrane and pyrenoid organization. Chloroplast ribosome levels rise rapidly when cells are transferred from acetate to minimal medium, whereas membrane reorganization occurs only after a lag. These results, combined with earlier studies of the photosynthetic properties of the mutant strain, suggest that proper membrane organization, Photosystem II activity, and ribulose-1,5-diphosphate carboxylase formation are dependent on the presence of chloroplast ribosomes. Other chloroplast components tested are unaffected by a 10-fold reduction in levels of chloroplast ribosomes.     

Sites of synthesis of chloroplast ribosomal proteins in Chlamydomonas

Cells of Chlamydomonas reinhardtii were pulse-labeled in vivo in the presence of inhibitors of cytoplasmic (anisomycin) or chloroplast (lincomycin) protein synthesis to ascertain the sites of synthesis of chloroplast ribosomal proteins. Fluorographs of the labeled proteins, resolved on two-dimensional (2-D) charge/SDS and one-dimensional (1-D) SDS-urea gradient gels, demonstrated that five to six of the large subunit proteins are products of chloroplast protein synthesis while 26 to 27 of the large subunit proteins are synthesized on cytoplasmic ribosomes. Similarly, 14 of 31 small subunit proteins are products of chloroplast protein synthesis, while the remainder are synthesized in the cytoplasm. The 20 ribosomal proteins shown to be made in the chloroplast of Chlamydomonas more than double the number of proteins known to be synthesized in the chloroplast of this alga.     

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

Euglena chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when ³⁵S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.     

Unassembled polypeptides of the plastidic ribosomes in heat-treated 70S-ribosome-deficient rye leaves

The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (M_r) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of M_r 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L polypeptide of large ribosomal subunit - M_r relative molecular mass - r-protein ribosomal polypeptide - S polypeptide of small ribosomal subunit - SDS sodium dodecyl sulfate     

The effects of salt stress on polypeptides in membrane fractions from barley roots

Cell fractions enriched in endoplasmic reticulum, tonoplast, plasma membrane, and cell walls were isolated from roots of barley (Hordeum vulgare L. cv CM 72) and the effect of NaCl on polypeptide levels was examined by two-dimensional (2D) polyacrylamide gel electrophoresis. The distribution of membranes on continuous sucrose gradients was not significantly affected by growing seedlings in the presence of NaCl; step gradients were used to isolate comparable membrane fractions from roots of control and salt-grown plants. The membrane and cell wall fractions each had distinctive polypeptide patterns on 2D gels. Silver-stained gels showed that salt stress caused increases or decreases in a number of polypeptides, but no unique polypeptides were induced by salt. The most striking change was an increase in protease resistant polypeptides with isoelectric points of 6.3 and 6.5 and molecular mass of 26 and 27 kilodaltons in the endoplasmic reticulum and tonoplast fractions. Fluorographs of 2D gels of the tonoplast, plasma membrane, and cell wall fractions isolated from roots of intact plants labeled with [³⁵S]methionine in vivo also showed that salt induced changes in the synthesis of a number of polypeptides. There was no obvious candidate for an integral membrane polypeptide that might correspond to a salt-induced sodium-proton anti-porter in the tonoplast membrane.     

The biosynthesis of ribulose bisphosphate carboxylase. Uncoupling of the synthesis of the large and small subunits in isolated soybean leaf cells

Isolated leaf cells from soybean (Glycine max) incorporate [35S]methionine into protein at a linear rate for at least 5h. Analysis of the products of incorporation by one-dimensional and two-dimensional polyacrylamide gel electrophoresis shows that major products are the large and small subunits of the chloroplast enzyme, ribulose bisphosphate carboxylase. The large subunit is synthesized by chloroplast ribosomes and the small subunit by cytoplasmic ribosomes. Addition of chloramphenicol to the cells reduces incorporation into the large subunit without affecting incorporation into the products of cytoplasmic ribosomes. Addition of cycloheximide or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide stops incorporation into the small subunit, but large subunit continues to be made for at least 4 h. For accurate estimates of incorporation into the large subunit, it is essential to use two-dimensional gel electrophoresis, because the large subunit region on one-dimensional gels is contaminated with the products of cytoplasmic ribosomes. Newly synthesized large subunits continue to enter complete molecules of ribulose bisphosphate carboxylase in the absence of small subunit synthesis. These results suggest that, in contrast to the situation in algal cells, the synthesis of the two subunits of ribulose bisphosphate carboxylase in the different subcellular compartments of higher plant cells is not tightly coupled over short time periods, and that a pool of small subunits exists in these cells. The results are disucssed in relation to possible mechanisms for the integration of the synthesis of the large and small subunits of ribulose bisphosphate carboxylase.     

Evidence for the nuclear location of the gene for chloroplast elongation factor G.

The chloroplast protein synthesis factor responsible for the translocation step of polypeptide synthesis on chloroplast ribosomes (chloroplast elongation factor G [EF-G]) has been detected in whole cell extracts and in isolated chloroplasts from Euglena gracilis. This factor can be detected by its ability to catalyze translocation on 70 S prokaryotic ribosomes such as those from E. coli. Chloroplast EF-G is present in low levels when Euglena is grown in the dark and can be induced more than 20-fold when the organism is grown in the light. The induction of this factor by light is inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. However, inhibitors of chloroplast protein synthesis such as streptomycin or spectinomycin have no effect on the induction of this factor by light. Furthermore, chloroplast EF-G can be partially induced by light in an aplastidic mutant (strain W₃BUL) which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-G resides in the nuclear genome, and that this protein is synthesized on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts.     

Synthesis of chloroplast membrane polypeptides during synchronous growth of Chlamydomonas reinhardtii

The synthesis of the major chloroplast membrane polypeptides has been studied during synchronous growth of Chlamydomonas reinhardtii. Under these conditions, chlorophyll is synthesized during the latter part of the light period and cell division takes place during the dark period. The profile of the chloroplast membrane polypeptides of C. reinhardtii has been well characterized and shown to contain two major classes by size (Hoober, J. 1970. J. Biol. Chem. 245:4327). Polypeptides of group I have a mol wt range of 50,000–55,000 daltons. The second region consists of at least three polypeptide groups, IIa, IIb, and IIc, having mol wt of 40,000, 31,000, and 27,000 daltons, respectively. The synthesis of these polypeptides has been measured using a double-labeling technique and a computer-aided statistical analysis. The rate of labeling of group I polypeptides is highest during the early light period and decreases after 6 h of growth. Group IIa is labeled from the beginning of the light period, but little synthesis of IIb occurs before 3 h, and significant amounts of label are not found in IIc before 5 h of growth. After approximately 8 h of light, groups IIb and IIc are synthesized at rates significantly greater than those of the other membrane polypeptides. The synthesis of the major polypeptide groups ceases in the dark. We conclude that the biosynthesis of the chloroplast membranes is a sequential or stepwise process.     

Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells

Techniques are described for the isolation of plastid thylakoid membranes from light-grown and dark-grown cells of Euglena gracilis var. bacillaris, and from mutants affecting plastid development. These membranes, which have minimal contamination with other cell fractions, are localized in sucrose gradients by using the thylakoid membrane sulfolipid as a specific marker. The plastid thylakoid membrane polypeptides isolated from these membranes were separated on SDS polyacrylamide gels and yielded patterns containing 30–40 polypeptides. Light-grown strain Z gave patterns identical with bacillaris. Since the plastid thylakoid polypeptide patterns obtained from dark-grown wild-type cells and from a bleached mutant W₃BUL in which plastid DNA is undetectable are identical, it appears that the proplastid thylakoid polypeptides of wild-type cannot be coded in plastid DNA and are probably coded in nuclear DNA. The plastid thylakoid polypeptide patterns obtained from various dark-grown mutants are identical to those obtained from dark-grown wild-type cells. Light-grown mutants, making large but abnormal chloroplasts, show a correlation between the amount of chlorophyll formed and the amount of a plastid thylakoid polypeptide thought to be associated with one of the pigment-protein light-harvesting complexes. Treatment with SAN 9789 (4-chloro-5-(methyl-amino)-2-(α,α,α,-trifluoro-m-tolyl)-3-(2H(pyridazinone) known to block carotenoid synthesis at the level of phytoene, causes a progressive loss of all plastid thylakoid polypeptides during growth in darkness and results in the establishment of a new, lower steady-state level of sulfolipid. At least ten of the plastid thylakoid polypeptides become labeled when isolated chloroplasts are supplied with radioactive amino acids; of these six are undectable in W₃BUL and are, therefore, candidates for coding by plastid DNA.     

Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes.

The purpose of this study was to investigate the contribution of mitochondrial and cytoplasmic protein synthesis to the biogenesis of cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase EC 1.9.3.1) and rutamycin-sensitive adenosine triphosphatase (ATP phosphohydrolase EC 3.6.1.3) in cultured oocytes of the toad, Xenopus laevis. X. laevis cytochrome oxidase was purified over 23-fold with respect to specific activity and over 29-fold with respect to specific heme a content from oocyte submitochondrial particles. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate separated the enzyme into six subunits with molecular weights of 44,000, 33,000, 23,000, 17,000, 12,000 and 9,500. the synthesis of the three larger subunits is sensitive to chloramphenicol (an inhibitor of mitochondrial protein synthesis), indicating that these subunits are made on mitochondrial ribosomes; the synthesis of the three smaller subunits is sensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and therefore occurs on cytoplasmic ribosomes. X. laevis rutamycin-sensitive ATPase, purified over 19-fold from oocyte submitochondrial pparticles, consists of 10 subunits with molecular weights of 56,000, 53,000, 41,000, 32,000, 29,000, 24,000, 21,000, 17,500 (2), and 11,500 on sodium dodecyl sulfate-polyacrylamide gels. The 29,000, 21,000, and one of the 17,500-dalton polypeptides are synthesized in the presence of cycloheximide and are, therefore, products of mitochondrial protein synthesis; the synthesis of the remaining seven subunits occurs in the presence of chloramphenicol, indicating that these subunits are made on cytoplasmic ribosomes. The synthesis of protein by mitochondria in cultured oocytes appears to be dependent upon cytoplasmic protein synthesis. In the presence of cycloheximide, the mitoribosomal synthesis of the subunits of cytochrome oxidase and rutamycin-sensitive ATPase is detectable only after a prior inhibition of mitochondrial protein synthesis by chloramphenicol. Oocyte mitochondrial ribosomes synthesize at least nine polypeptides after chloramphenicol treatment, three of which are components of neither cytochrome oxidase nor rutamycin-sensitive ATPase.