site of synthesis of the proteins of mammalian mitochondrial

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

VOl. 260, No . 10, Issue of May 25, pp. 63676372,1985 Printed in U. S. A .

Site of Synthesis of the Proteins of Mammalian Mitochondrial Ribosomes EVIDENCE FROM CULTURED BOVINE CELLS*

(Received for publication, July 23,1984)

Gretchen L. SchieberS and Thomas W. O’BrienQ From the Department of Biochemistry and Molecular Biology, Uniuersity of Florida, College of Medicine, J . Hillis Miller Health Center, Gainesuille, Florida 32610

In order to determine the sites of synthesis of the proteins of the mammalian mitochondrial ribosome (mitoribosome), bovine (MDBK) cells were labeled with [35S]methionine in the presence of inhibitors of mitochondrial and cytoplasmic protein synthesis. La- beling in the absence of cytoplasmic protein synthesis produced a “blank” fluorogram, indicating that there is no mitochondrial product. Additionally, incorporation of [3SS]methionine into the enumerated mitoribo- soma1 proteins continued in the absence of mitochondrial protein synthesis. Finally, it was demonstrated that mitoribosomal proteins can be both translated and assembled into complete mitoribosomes in the absence of mitochondrial protein synthesis. These results indicate that in mammals, as opposed to lower eukaryotes, all of the mitoribosomal proteins are products of cytoplasmic protein synthesis.

While mitochondria do contain their own DNA and a complete protein synthesis system, including ribosomes, tRNA, rRNA, and all of the accessory proteins, they synthesize only a few of the several hundred proteins necessary for proper mitochondrial function. The vast majority of mitochondrial proteins are translated on cytoplasmic (80 S) ribosomes and imported into mitochondria. These imported proteins must include most of the proteins of the mitochondrial ribosome (mitoribosome’), but it has been shown in two lower eukaryotes that one of the mitoribosomal proteins is a product of mitochondrial protein synthesis (var 1 in yeast (1, 2) and S-5 in Neurospora (3)). This small subunit protein appears to serve as a rRNA or protein assembly site during the synthesis of the subunit (4-6), since impairing its synthesis results in incomplete assembly of these subunits. A similar protein has not been identified in any other system.

In recent years, the mitochondrial DNA in several mammalian species (7-11) has been completely sequenced. This information confirmed the sequences of mitochondrial rRNA, tRNA, and five previously identified products of mitochon-

* This work was supported by United States Public Health Service Grants GM-15438 and GM-25888. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Current address: Laboratory of Cellular Biology and Genetics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Bethesda, MD 20205.

§ To whom all correspondence should be addressed. ’ The abbreviations used are: mitoribosome, mitochondrial ribo-

some; monosome, monomeric ribosome; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis,

drial protein synthesis. Eight additional unidentified reading frames present in the mammalian genomes were also discov- ered. While the sequences of the putative proteins coded for by these regions are known and some translation has been demonstrated (12-14), matching these presumptive genes with corresponding functional mitochondrial proteins has been difficult. The presence of a mitochondrially produced, mitoribosomal protein in lower eukaryotes implied that one of the unidentified reading frames in mammalian systems might code for an analogous protein.

The research reported in this paper examines the site of synthesis of the mitoribosomal proteins in a mammalian system. Bovine cells in culture were studied using specific inhibitors of cytoplasmic and mitochondrial protein synthesis to identify which of the mitoribosomal proteins are synthesized in each compartment. The results from this technique indicate that all of the proteins of the mammalian mitoribosome are cytoplasmically synthesized.

EXPERIMENTAL PROCEDURES AND RESULTS~

Labeling of Mitoribosomal Proteins Is Eliminated in the Absence of Cytoplasmic Protein Synthesis-In order to inhibit labeling of the products of cytoplasmic protein synthesis, cultured bovine (MDBK) cells were labeled in the presence of cycloheximide (Fig. 4A). With cycloheximide, only products of mitochondrial protein synthesis should incorporate radioactivity. During the chase period, in the absence of both radioactivity and the inhibitor, any components required for assembly of ribosomes can be synthesized and the particles should assemble, but components made by the nucleo-cytoplasmic system will not be radiolabeled. The mitoribosomal subunits were prepared using a single sucrose density gradient and were analyzed by two-dimensional PAGE. No spots are observed on fluorograms from either large or small ribosomal subunits labeled in the presence of cycloheximide (Fig. 1, A and B ) . In contrast, the subunit preparations from parallel control cells (Fig. 1, C and D) show a great deal of incorporated radioactivity. The prominent spots in these fluorograms are contaminating mitochondrial proteins which appear because the subunits were prepared by the single gradient method. The known proteins of the mitoribosome are visible as paler

* Portions of this paper (including “Experimental Procedures,” part of “Results,” and Figs. 4-7) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard

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6368 Site of Synthesis of Mammalian Mitoribosomal Proteins

FIG. 1 . Fluorograms of mitoribosomal subunits from cells labeled in the absence of cytoplasmic protein synthesis. Mito- chondrial ribosomal subunits lrom cells labeled in the presence of cycloheximide ( 3 0 0 pg/ml) were analyzed by two-dimensional PAGE and fluorography. Fluorograms of the small ( A ) and large ( H ) subunits are shown. Hihosomal subunits from cells labeled in parallel in the absence of inhibitors are shown in c‘ (28 S) and 1) (39 S).

spots. This experiment was performed twice, giving the same result b0t.h times.

Thus, conditions of the labeling period were adequate for the incorporation of [““Slmethionine into mitochondrial ribosomal proteins, but that incorporation is blocked by cycloheximide.

Mitoribosomal Proteins Are Synthesized and Assemble Nor- ma1I.v in the Absence of Mitochondrial Protein Synthesis- Mitochondrial protein synthesis was inhibited with chloramphenicol in two protocols. In the first, a converse experiment to the one described above, MDRK cells were labeled with [““Slmethionine in the presence of chloramphenicol, allowing incorporation only into those proteins produced by the nucleo- cytoplasmic system (Fig. 4R). As before, proteins made during the chase period (by the mitochondrial protein synthetic syst.em) should not have any incorporated radioactivity. Since this inhibitor has a low toxicity to these cells, the labeling period could be much longer (8-16 h), producing ribosomes of higher specific activity. This allowed pure ribosomal subunits t,o be prepared using two sucrose density gradients (see “Ex- perimental Procedures”) before two-dimensional PAGE analysis.

The second protocol for labeling cells in the presence of chloramphenicol was designed to test a prediction. That is, if a11 of the mitoribosomal proteins are c.ytoplasmically synthesized (as indicated by the absence of incorporated label in the presence of cycloheximide), then it should be possible to completely synthesize and assemble mitoribosomes in the absence of mitochondrial protein synthesis. In this protocol

(Fig. 4C) after the chloramphenicol is added, the cells are preincubated for 2 h to allow existing mitochondrial protein synthetic products to be utilized. Also, there is no chase period; chloramphenicol is kept in the solutions until the end of the harvesting procecure.

As seen in Figs. 2 (28 S subunit) and 3 (39 S subunit), the proteins of mitochondrial ribosomes are synthesized and assembled normally in the absence of mitochondrial protein synthesis. The patterns of mitoribosomal proteins in these fluorograms correspond to the characteristic patterns of the carrier bovine liver mitoribosomal proteins revealed by Coo- massie Blue stain (shown schematically in A ) . Because of some variability in recovery and intensity of specific proteins, each of these protocols was performed 3 times. Most of the enumerated proteins of the bovine mitoribosome were consistently labeled in the presence of chloramphenicol both with ( H ) and without (C) a chase period (shown by f i l l d spots in the schematic diagrams) and in the absence of inhibitors ( I ) ) . One of the major differences between the stained map and the fluorograms is a group of proteins (S26, 1~13, L24, LXj, L34, L39, and L40) which never incorporated any radiolabel. They probably do not contain methionine, and must, therefore, be products of cytoplasmic protein synthesis (see “Dis- cussion”).

Some of the mitoribosomal proteins are variable in appearance in this sytem ofanalysis (15) . For example, some proteins (S9, S12, S13, S25, L27, and L45), were not labeled in most experiments, but seemed co-migratory or nearly co-migratory with a radioactive spot in at least one experiment. Impor- tantly, when they were labeled, these proteins labeled in the pattern expected for products of cytoplasmic protein synthesis. An additional group of proteins (S2, !%, S11, SI;. S19,

A ’ - .*I . .,, . ..,

0 - -8

B

FIG. 2. 28 S mitoribosomal subunits from cells laheled in the absence of mitochondrial protein synthesis. The schematic diagram ( A ) shows as /i//ed c,trc/rs those proteins o f the small mitorihosomal subunit which consistently incorporated radiolaheled methionine as detected in fluorograms of subunits from cell laheled in the presence of chloramphenicol both with ( H ) and without ( ( ‘ I a chase period and in the control (no inhihitor. I ) ) . ’The small srhunit proteins which did not incorporate the radioactive amino acid in some or all experiments are shown as dotted c i r c h (see text j .

Site of Synthesis of Mammalian Mitoribosomal I’rot&s 6369

C D

0 0

FIG. :I. 39 S mitoribosomal subunits from cells labeled in the absence of mitochondrial protein synthesis. The schematic diagram ( A ) shows as / i l lrd circkrs those proteins of‘ the large mitorihosomal suhunit which consistentlv incorporated radiolaheled methionine as detected in fluorograms of subunits from cells laheled in the presence of‘ chloramphenicol hoth with (8) and without ( ( 7 a chase period and in the control ( n o inhihitor. [ I ) . The large suhunit proteins which did not incorporate the radioactive amino acid in some or all experiments are shown as doftrd circlrs (see text).

and S20) did not always appear on the electropherograms, as judged by the Coomassie Blue stain pattern of the carrier, hut, when present, were labeled. In the fluorogram shown in Fig. :VI, prot.eins L49, L50, L51, and L52 are not observed simply because they were lost during the first electrophoretic dimension. They are easily observed in the other fluorograms shown, however, and are grouped with those proteins which always incorporated radiolabel. Two proteins, S24 and L.76, did not, appear on any of the electrophoretic separations used in these experiments (see “Discussion”). Other cytoplasmically synthesized proteins appeared irreproducibly in the fluorograms. Some of these are caused by subunit cross-contamination, while others are probably nonribosomal. These experiment.s demonstrate that the enumerated proteins of mammalian mitoribosomes are synthesized in the presence of chloramphenicol. Additionally, correctly assembled, properly sedimenting subunits are produced entirely in the absence of mitochondrial protein synthesis.

DISCUSSION

T h i s work addresses the question of whether or not any of t,he mitoribosomal proteins are products of mitochondrial protein synthesis. Ry using specific inhibitors, mitochondrial and cytoplasmic protein synthesis were “isolated” and studied in an intact cellular system, bovine tissue culture cells (MDRK cells). The proteins of mammalian mitoribosomes labeled in a manner expected for products of cytoplasmic protein synthesis. That is, they fail to label in the presence of cycloheximide and do incorporate label in the presence of chloramphenicol. Furthermore, correctly sedimenting ribosomal subunits are synthesized completely in the presence of

chloramphenicol, and the analysis of their proteins indicates normal assembly of mitoribosomes even in the absence of mitochondrial protein svnthesis.

This experimental system offers several advantages. First ly. there is a built-in selection criterion for mitoribosomal proteins. Since a complete spectrum of specific antibodies does not exist, mitoribosomal proteins are selected by allowing the cells sufficient t,ime, either in the absence or presence o f inhibitors, to assemtde proteins into mitorihosomal particles. Secondly, bovine liver mitoribosomes are easily obtained for protein carrier. Added at mitochondrial lysis. after the ribosomes are labeled and assembled, they add needed bulk t o the material being isolated. This makes the manipulations easier and is particularly important in reducing handling losses. During the purification of ribosomes. i t is the A,,1, measure- ment of the carrier which is used to judge fractions to be pooled and tot.al recovery. Thus, by definition, mitoribosomal proteins are those from the gradient positions fnr intact, fully assembled mitoribosomal subunits. During t he electrophoretic analysis, the well-characterized carrier pattern (1.5) shows the correct position for the ribosomal proteins, and exact co- migration can be used to identify mitoribosomal proteins and distinguish contamination.

In analyzing these results, several factors must be considered. The blank fluorograms which resulted with the inhihitor cycloheximide would also have been produced i f t he conditions for the labeling period (media, length of incubation, specific activity of radiotracer, etc.) or for recovery and analysis of ribosomal subunits (mitochondrial isolation, length of fluorography, et.c.) were inadequate to visualize incorporation into mitoribosomal proteins. A parallel control experiment, however, demonst.rated that the synthesis of mitorit)osomal proteins was observable under these conditions. Additionally, during prolonged incubations in the absence of cytoplasmic synthesis, mitochondrial protein synthesis becomes inhibited and eventually mitochondrial products are degraded (16, 17). In order to ensure that the cycloheximide labeling condition was not excessive, intact mitochondria from these cells were examined and showed the expected limited number of mitochondrially synthesized protein bands, demonstrating that the products of mitochondrial protein svnthesis were translated during the incubation period and that they would be available for assembly into their correct functional sites during the chase period. However, a mitoribosomal protein might be a minor product, not discernible by one-dimensional SI)S- PAGE analysis. Additionally, sequestration in the membrane might give added protection from proteolysis to electron transport proteins but not to a ribosomal protein. Since the effect of these factors cannot be evaluated, the blank fluorograms are not conclusive data.

Another important factor is the effectiveness of chloramphenicol inhibition in these cells. In these experiments, the analysis examines only those proteins which assemble into properly sedimenting mitoribosomes. Hecause of the small amount of material available, the results would not d i s t inn~ish unaffected incorporation from partial incorporation caused by incomplete chloramphenicol inhibition. We have shown. however, that chloramphenicol reduces mitochondrial protein synthesis below detectable levels even at 1 0 0 pg/ml.

All of these experiments require high specific activity ”S- labeled amino acids (either methionine or cvsteine) for d e - quat.e detection. Fortunatelv, since the amino acid sequence of all possible mitochondrial products can be predicted from the known DNA sequence (9). it appears that all mitnchon- drial protein synthetic products contain a high proportion of methionine residues. Those proteins (c .g l,l:{) missing on all

6370 Site of Synthesis of Mammalian Mitoribosomal Proteins

fluorograms of mitoribosomes probably simply lack methionine residues, and therefore, cannot be mitochondrial products.

Finally, this analysis is based upon the ability to detect all of the mitoribosomal proteins by a specific system of two- dimensional PAGE. Yet, a few of the mitoribosomal proteins are variable in their appearance in this system of analysis (15). Additionally, the possibility must be considered that there are ribosomal proteins that are never detected in the two-dimensional PAGE analysis (a protein co-migratory with another protein or one not solubilized by the extraction conditions). However, the subunits examined had migrated as assembled subunits in sucrose density gradients. If chloramphenicol blocks mitoribosome synthesis at a precursor stage, such particles would be completed with unlabeled proteins during the chase period and would be deficiently labeled in those proteins which were added after any mitochondrial product. Importantly, this protocol produced the same pattern of spot intensities as the parallel control.

However, if proteins made cytoplasmically during the labeling period were still available for assembly during the chase period (proteins which required several minutes for import into the mitochondria or unassembled proteins inside the organelle that are not quickly degraded), then, when the inhibition is released, a mitochondrially synthesized protein could be produced and assembly could continue. Even without a chase period, mitoribosomes from cells labeled and assembled in the presence of chloramphenicol show the same patterns as their uninhibited counterparts. This indicates that the absence of mitochondrial protein synthesis did not block synthesis or assembly of both mitoribosomal subunits.

I t is known that in yeast and Neurospora, the small ribosomal subunits formed in the absence of mitochondrial protein synthesis are defective. They migrate incorrectly in sedi- mentation analysis, and they specifically lack a few proteins which assemble after the mitochondrial product. These experiments have demonstrated that no protein analogous to either S-5 in Neurospora or var 1 in yeast is present in mammalian mitoribosomes. These data do not completely rule out the possibility of “hidden” mitoribosomal proteins, proteins which were not detected either because they are not present in the electropherogram or because they are not distinguished from another similarly migrating protein. How- ever, if such a mitoribosomal protein does exist, it does not affect the ability of any other protein to assemble onto the mitoribosome, and, therefore, is functioning in some manner which differs from the behaviur of the mitochondrially produced protein of the mitoribosomes in lower eukaryotes.

These experiments indicate that in mammals, as opposed to the lower eukaryotes, all of the proteins of the mitoribosome are cytoplasmically synthesized. The only other known example of a change in mitochondrial products is the “dicyclo- hexylcarbodiimide-binding protein” of the F1 ATPase, which is made mitochondrially in yeast and cytoplasmically in Neu- rospora and mammals (9,18,19). If, indeed, the mitochondrial product protein in lower eukaryotes functions in ribosome assembly, that function is performed in mammals by a protein or proteins imported from the cytoplasm. It might be that a

protein of identical function is imported. Because of the differences in the structure of the two classes of mitoribosomes, it is also possible that the ribosomal domain involved is sufficiently changed in mammals that a different assembly mechanism is used. Considering the highly proteinaceous nature of the mammalian mitoribosome, perhaps this is not so surprising. I t might be that the ribosomal region of S-5 or var 1 in lower eukaryotes is functionally the same in mammalian mitoribosomes, but structurally completely different.

The data presented here demonstrate that mitochondria are not obligated to make a set of specific types of proteins, but that the protein synthetic products are evolving in re- sponse to, or in partnership with, the proteins encoded on the nuclear genome.

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6372 Site of Synthesis of Mammalian Mitoribosomal T'rotcins

site of synthesis of the proteins of mammalian mitochondrial

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