The cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria

The mechanism of import of proteins into mitochondria was studied by using the peptide of the presequence of ornithine aminotransferase (the extrapeptide), which was chemically synthesized and is composed of 34 amino acids. When the extrapeptide was incubated with isolated mitochondria in the presence of a rabbit reticulocyte lysate at 25 degrees C, it was imported into the mitochondrial matrix, and the import depended on the inner membrane potential, but not added ATP. The import of several precursors of mitochondrial proteins was competitively inhibited by the presence of excess extrapeptide in the reaction system, indicating that the extrapeptide and mitochondrial proteins were imported by the same machinery. Import of the extrapeptide was significantly stimulated by addition of a rabbit reticulocyte lysate, and a component of the lysate (the cytosolic factor) stimulating import of the extrapeptide was purified about 20,000 times by successive column chromatography on DEAE-cellulose and aminopentyl-Sepharose 4B. The binding of the extrapeptide to liposomes composed of egg lecithin and partially purified receptor of the precursor of mitochondrial protein (Ono, H., and Tuboi, S., (1985) Biochem. Int. 10, 351-357) required the cytosolic factor when the concentration of the peptide was less than 1.5 X 10(-8) M, suggesting that the physiological binding of the precursors of mitochondrial proteins to the receptor is dependent on the cytosolic factor. The extrapeptide and the cytosolic factor were shown to form a complex. From these results, the mechanism of binding of the extrapeptide to the receptor of the mitochondrial outer membrane is suggested to be as follows: the peptide (the precursor of mitochondrial protein) and the cytosolic factor form a complex, and then the complex is recognized by and bound to the receptor.     

Presequence binding factor-dependent and -independent import of proteins into mitochondria.

A cytosolic protein factor(s) is involved in the import of precursor proteins into mitochondria. PBF (presequence binding factor) is a protein factor which binds to the precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) but not to the mature OTC, and is required for the mitochondrial import of pOTC. The precursors for aspartate aminotransferase and malate dehydrogenase as well as pOTC synthesized in a reticulocyte lysate were efficiently imported into the mitochondria. However, the precursors synthesized in the lysate depleted for PBF by treatment with pOTC-Sepharose were not imported. Readdition of the purified PBF to the depleted lysate fully restored the import. pOTC synthesized in the untreated lysate sedimented as a complex with a broad peak of around 9 S, whereas pOTC synthesized in the PBF-depleted lysate sedimented at an expected position of monomer (2.5 S). When the purified PBF was readded to the depleted lysate, pOTC sedimented as a complex of about 7 S. In contrast to most mitochondrial proteins, rat 3-oxoacyl-CoA thiolase is synthesized with no cleavable presequence and an NH2-terminal portion of the mature protein functions as a mitochondrial import signal. The thiolase synthesized in the PBF-depleted lysate could be efficiently imported into the mitochondria, and readdition of PBF had little effect on the import. The thiolase synthesized in the untreated, the PBF-depleted, or the PBF-readded lysate sedimented at an expected position of monomer (2.5 S). These observations provide support for the existence of PBF-dependent and -independent pathways of mitochondrial protein import.     

Mitochondrial precursor protein. Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence

A hybrid precursor protein constructed by fusing the mitochondrial matrix-targeting signal of rat preornithine carbamyl transferase to murine cytosolic dihydrofolate reductase (designated pO-DHFR) was expressed in Escherichia coli. Following purification under denaturing conditions, pO-DHFR was capable of membrane translocation when diluted directly into import medium containing purified mitochondria but lacking cytosolic extracts. This import competence was lost with time, however, when the precursor was diluted and preincubated in medium lacking mitochondria, unless cytosolic proteins (provided by rabbit reticulocyte lysate) were present. Identical results were obtained for purified precursor made by in vitro translation. The ability of the cytosolic proteins to maintain the purified precursor in an import-competent state was sensitive to protease, N-ethylmaleimide (NEM), and was heat labile. Further, this activity appeared to be signal sequence dependent. ATP was not required for the maintenance of pO-DHFR competence, nor did purified 70-kDa heat shock protein (the constitutive form of Hsp70) substitute for this activity. Interestingly, however, purified Hsp70 prevented aggregation of the precursor in an ATP-dependent manner and, as well, retarded the apparent rate and extent of pO-DHFR folding. Partial purification of reticulocyte lysate proteins indicated that competence activity resides within a large mass protein fraction (200-250 kDa) that contains Hsp70. Sucrose density gradient analysis revealed that pO-DHFR reversibly interacts with components of this fraction. Pretreatment of the fraction with NEM, however, significantly stabilized the subsequent formation of a complex with the precursor. The results indicate that Hsp70 can retard precursor polypeptide folding and prevent precursor aggregation; however, by itself, Hsp70 cannot confer import competence to pO-DHFR. Maintenance of import competence correlates with interactions between the precursor and an NEM-sensitive cytosolic protein fraction. Efficient dissociation of the precursor from this complex appears to require a reactive thiol moiety on the cytosolic protein(s).     

Investigations on the in vitro import ability of mitochondrial precursor proteins synthesized in wheat germ transcription-translation extract

Mitochondrial precursor proteins synthesized in rabbit reticulocyte lysate (RRL) are readily imported into mitochondria, whereas the same precursors synthesized in wheat germ extract (WGE) fail to be imported. We have investigated factors that render import incompetence from WGE. A precursor that does not require addition of extramitochondrial ATP for import, the F(A)d ATP synthase subunit, is imported from WGE. Import of chimeric constructs between precursors of the F(A)d protein and alternative oxidase (AOX) with switched presequences revealed that the mature domain of the F(A)d precursor defines the import competence in WGE as only the construct containing the presequence of AOX and mature portion of F(A)d (pAOX-mF(A)d) could be imported. Import competence of F(A)d and pAOX-mF(A)d correlated with solubility of these precursors in WGE, however, solubilization of import-incompetent precursors with urea did not restore import competence. Addition of RRL to WGE-synthesized precursors did not stimulate import but addition of WGE to the RRL-synthesized precursors or to the over-expressed mitochondrial precursor derived from the F1beta ATP synthase precursor inhibited import into mitochondria. The dual-targeted glutathione reductase precursor synthesized in WGE was imported into chloroplasts, but not into mitochondria. Antibodies against the 14-3-3 guidance complex characterized for chloroplast targeting were able to immunoprecipitate all of the precursors tested except the F(A)d ATP synthase precursor. Our results point to the conclusion that the import incompetence of WGE-synthesized mitochondrial precursors is not presequence dependent and is a result of interaction of WGE inhibitory factors with the mature portion of precursor proteins.     

The precursor to ornithine carbamyl transferase is transported to mitochondria as a 5S complex containing an import factor

The precursor to ornithine carbamyl transferase (Mr = 40,000) was synthesized in a rabbit reticulocyte lysate system and purified by immunoaffinity chromatography. Import of purified precursor by isolated mitochondria depended upon the presence of import factor(s) in fresh reticulocyte lysate. Velocity sedimentation analyses indicated that import factor binds to precursor to form a 5S complex (approximately 90 kDa); in this form, precursor was efficiently imported by isolated mitochondria. The ability of the 5S complex to deliver precursor into mitochondria was not affected by pretreatment with high concentrations of RNase. Import factor did not bind to mitochondria in the absence of precursor; upon binding of precursor to mitochondria in the presence of import factor, subsequent transmembrane uptake of precursor did not require the continued presence of additional lysate components.     

Reinvestigation of the requirement of cytosolic ATP for mitochondrial protein import

Protein import into mitochondria requires the energy of ATP hydrolysis inside and/or outside mitochondria. Although the role of ATP in the mitochondrial matrix in mitochondrial protein import has been extensively studied, the role of ATP outside mitochondria (external ATP) remains only poorly characterized. Here we developed a protocol for depletion of external ATP without significantly reducing the import competence of precursor proteins synthesized in vitro with reticulocyte lysate. We tested the effects of external ATP on the import of various precursor proteins into isolated yeast mitochondria. We found that external ATP is required for maintenance of the import competence of mitochondrial precursor proteins but that, once they bind to mitochondria, the subsequent translocation of presequence-containing proteins, but not the ADP/ATP carrier, proceeds independently of external ATP. Because depletion of cytosolic Hsp70 led to a decrease in the import competence of mitochondrial precursor proteins, external ATP is likely utilized by cytosolic Hsp70. In contrast, the ADP/ATP carrier requires external ATP for efficient import into mitochondria even after binding to mitochondria, a situation that is only partly attributed to cytosolic Hsp70.     

Import of chemically synthesized signal peptides into rat liver mitochondria

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.     

The requirement of heat shock cognate 70 protein for mitochondrial import varies among precursor proteins and depends on precursor length.

The cytosolic heat shock cognate 70-kDa protein (hsc70) is required for efficient import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria (K. Terada, K. Ohtsuka, N. Imamoto, Y. Yoneda, and M. Mori, Mol. Cell. Biol. 15:3708-3713, 1995). The requirement of hsc70 for mitochondrial import of various precursor proteins and truncated pOTCs was studied by using an in vitro translation import system in which hsc70 was completely depleted. hsc70-dependent import of pOTC was about 60% of the total import, while import of the aspartate aminotransferase precursor, the serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase was about 50, 30, and 0%, respectively. The subunit sizes of these four precursor proteins were 40 to 47 kDa. When pOTC was serially truncated from the COOH terminal, the hsc70 requirement decreased gradually and was not evident for the shortest truncated pOTCs of 90 and 72 residues. These truncated pOTCs were imported and proteolytically processed rapidly in 0.5 to 2 min at 25 degrees C, and the processed mature portions and the presequence portion were rapidly degraded. Sucrose gradient centrifugation analysis followed by import assay showed that pOTC synthesized in rabbit reticulocyte lysate forms an import-competent complex of about 11S in an hsc70-dependent manner. S values of import-competent forms of aspartate aminotransferase precursor, serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase were 9S, 9S, and 4S, respectively. Thus, the S value decreased as the hsc70 dependency decreased. Precursor proteins were coimmunoprecipitated from the reticulocyte lysate containing the newly synthesized precursor proteins with an hsc70 antibody. The amount of coimmunoprecipitated proteins was much larger in the absence of ATP than in its presence. Among the four precursor proteins, the amount of coimmunoprecipitated protein decreased as the hsc70 dependency decreased.     

Membrane and cytosolic components affecting transport of the precursor for ornithine carbamyltransferase into mitochondria

A higher molecular weight precursor (Mr = 39,000) to the liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = 36,000), is imported and processed by heart mitochondria in vitro in a manner similar to liver mitochondria. In both systems, however, an additional 37-kDa ornithine carbamyltransferase polypeptide appears, but this arises from nonspecific events and, therefore, does not represent a bona fide intermediate in the overall processing sequence. Our experiments demonstrate that the outer mitochondrial membrane of mitochondria contains a protease-sensitive (5 micrograms of trypsin or chymotrypsin/ml, 15 min at 2 degrees C), salt-resistant (1.0 M KCl) protein which is required to maintain import functions. In addition, functional post-translational import requires a component of the reticulocyte lysate (i.e. cytosol) that is used for initially synthesizing precursor enzyme. The component is retained by Sephadex G-25. Import of Sephadex G-25-excluded precursor is restored by fresh reticulocyte lysate but not by a combination of other additives, including Mg2+, K+, ATP, ADP, Pi, succinate, and total translation mixture (minus lysate).     

A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.

Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2-terminal 'leader' peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26-K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti-DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti-DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell-free system and incubated post-translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post-translational import of a chimeric precursor protein by mitochondria.     

Targeting of a chemically pure preprotein to mitochondria does not require the addition of a cytosolic signal recognition factor.

To analyze the role of cytosolic cofactors in mitochondrial protein targeting, we prepared a chemically pure mitochondrial preprotein. When diluted out of 7 M urea, this precursor protein was efficiently imported into mitochondria without the addition of cytosolic cofactors. Extensive prewashing of mitochondria (up to 2 M KCl) did not reduce its import. Import of the purified precursor showed the characteristics of authentic mitochondrial import including use of the receptor MOM19, requirement for a membrane potential, and proteolytic processing. When the precursor was preincubated at a low concentration of urea, cytosolic cofactors were needed to preserve its import competence. We conclude that targeting of this preprotein via the mitochondrial master receptor MOM19 does not require a cytosolic signal recognition factor; cytosolic cofactors apparently have chaperone-like functions in mitochondrial protein uptake. Moreover, we found that a cleavable presequence was sufficient to direct protein import via MOM19. Together with the cofactor-independent function of MOM19, it is thus conceivable that MOM19 functions as mitochondrial presequence receptor.     

Transport of ornithine carbamoyltransferase precursor into mitochondria. Stimulation by potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s)

The precursor of rat liver ornithine carbamoyltransferase (EC 2.1.3.3) synthesized in vitro was taken up and processed to the mature enzyme by isolated rat liver mitochondria. Potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s), in addition to the precursor and the mitochondria, were required for maximal transport and processing of the precursor. The concentrations of potassium and magnesium ions required for maximal transport and processing were about 120 and 0.8-1.6 mM, respectively. Dialyzed postribosomal supernatant of rabbit reticulocyte lysate (36 mg of protein/ml), in combination with potassium and magnesium ions, stimulated the transport and processing severalfold. The stimulatory activity of the dialyzed lysate was inactivated by trypsin treatment or heating at 100 degrees C for 2 min. No significant amount of the precursor was associated with the mitochondria when incubation was performed in the absence of these components. These results suggest that potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s) stimulate the binding and transport of the ornithine carbamoyltransferase precursor to the mitochondria. Dialyzed supernatant of rabbit erythrocyte lysate was equally effective in stimulating the precursor transport and processing, and a dialyzed cytosol fraction of Ehrlich ascites cells was partly stimulatory. On the other hand, dialyzed cytosol fractions of rat liver and rat kidney, and dialyzed supernatant of wheat germ extracts did not stimulate the precursor transport and processing but rather inhibited it.     

A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites

Bovine pancreatic trypsin inhibitor (which contains three intramolecular disulfide bridges) was chemically coupled to the COOH terminus of a purified artificial mitochondrial precursor protein. When the resulting chimeric precursor was presented to energized isolated yeast mitochondria, its trypsin inhibitor moiety prevented the protein from completely entering the organelle; the protein remained stuck across both mitochondrial membranes, with its NH2 terminus in the matrix and its trypsin inhibitor moiety still exposed on the mitochondrial surface. The incompletely imported protein appeared to "jam" mitochondrial protein import sites since it blocked import of three authentic mitochondrial precursor proteins; it did not collapse the potential across the mitochondrial inner membrane. Quantification of the inhibition indicated that each isolated mitochondrial particle contains between 10(2) and 10(3) protein import sites.     

14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants

Transit sequences of chloroplast-destined precursor proteins are phosphorylated on a serine or threonine residue. The amino acid motif around the phosphorylation site is related to the phosphopeptide binding motif for 14-3-3 proteins. Plant 14-3-3 proteins interact specifically with wheat germ lysate-synthesized chloroplast precursor proteins and require an intact phosphorylation motif within the transit sequence. Chloroplast precursor proteins do not interact with 14-3-3 when synthesized in the heterologous reticulocyte lysate. In contrast, a precursor protein destined for plant mitochondria was found to be associated with 14-3-3 proteins present in the reticulocyte lysate but not with 14-3-3 from wheat germ lysate. This indicates an unrecognized selectivity of 14-3-3 proteins for precursors from mitochondria and plastids in plants in comparison to fungi and animals. The heterooligomeric complex has an apparent size of 200 kD. In addition to the precursor protein, it contains 14-3-3 (probably as a dimer) and a heat shock protein Hsp70 isoform. Dissociation of the precursor complex requires ATP. Protein import experiments of precursor from the oligomeric complex into intact pea chloroplasts reveal three- to fourfold higher translocation rates compared with the free precursor, which is not complexed. We conclude that the 14-3-3-Hsp70-precursor protein complex is a bona fide intermediate in the in vivo protein import pathway in plants.     

A synthetic signal peptide blocks import of precursor proteins destined for the mitochondrial inner membrane or matrix

A peptide corresponding to amino acids 1-27 of preornithine carbamyltransferase (pOCT) has been chemically synthesized. When added to energized mitochondria in vitro, 20 microM of the peptide, designated pO(1-27), resulted in a collapse of the electrochemical potential across the mitochondrial inner membrane. This effect on transmembrane potential was not observed, however, when pO(1-27) was added to energized mitochondria under conditions that support in vitro import of precursor proteins (i.e. in the presence of reticulocyte lysate). The latter finding, therefore, made possible an examination of the ability of pO(1-27) to block import of homologous and heterologous proteins into the organelle. At 5-10 microM, pO(1-27) prevented import of pOCT in vitro; inhibition was overcome by increasing the concentration of pOCT. In contrast, pO(16-27), a peptide corresponding to amino acids 16-27 of pOCT and exhibiting a charge:mass ratio similar to pO(1-27) had no such inhibitory effect. pO(1-27) blocked import of other unrelated precursor proteins destined either for the mitochondrial matrix (pre-malate dehydrogenase and a hybrid protein containing the signal sequence of pre-carbamyl phosphate synthetase) or for the mitochondrial inner membrane (pre-thermogenin).     

Protein import into yeast mitochondria is inhibited by antibodies raised against 45-kd proteins of the outer membrane.

Import of several precursor proteins into isolated yeast mitochondria is inhibited by rabbit antiserum raised against the total mitochondrial outer membrane or against electrophoretically purified 45-kd outer membrane proteins. Antisera against other outer membrane proteins are only marginally active or inactive. Inhibition by the antiserum against 45-kd proteins is only weak with untreated mitochondria, but reaches 80-90% with mitochondria that had been pretreated with 0.1 mg/ml trypsin. This trypsin pretreatment by itself inhibits precursor import only slightly (30-50%). Selective inhibition of import does not correlate with binding of the various IgGs to the mitochondrial surface and is also observed with the corresponding Fab fragments. Inhibition by antibodies against 45-kd outer membrane proteins strongly suggests the existence of a mitochondrial surface protein mediating protein import and offers a means of isolating this protein.     

ADP-ATP carrier of Saccharomyces cerevisiae contains a mitochondrial import signal between amino acids 72 and 111

The ADP-ATP carrier (also referred to as the adenine nucleotide translocator) of Saccharomyces cerevisiae is encoded by a nuclear gene, translated in the cytosol, and imported into the mitochondrial inner membrane. In order to study the determinants of mitochondrial import, a series of fusion proteins, consisting of the first 21, 72, and 111 amino acids of the ADP-ATP carrier, joined to mouse dihydrofolate reductase were generated. Dihydrofate reductase is a cytoslic protein that does not bind mitochondria. The reticulocyte lysate reaction containing the 35S-methionine-labeled protein was incubated with mitochondria in a buffer containing 3% BSA. Following incubation for import, the reactions were treated with 1 mM PMSF or 25 micrograms/ml proteinase K; mitochondria were reisolated and analyzed by gel electrophoresis. The 21 and 72 amino acid hybrid proteins showed a low level of binding to mitochondria: the bound form was entirely protease accessible. The 111 amino acid hybrid protein was imported to a protease-protected location within mitochondria. It is concluded that the first 72 amino acids of the ADP-ATP carrier do not suffice to import the protein into mitochondria and that the region between amino acids 72 and 111, a region that contains a transmembrane-spanning domain, constitutes at least part of the mitochondrial import signal.     

Protein import into mitochondria in a homologous yeast in vitro system

To study the import of proteins into mitochondria we developed a homologous in vitro system in which mitochondria and cell-free translation extract are both derived from the yeast Saccharomyces cerevisiae. This system allows the synthesis of precursor proteins in the presence of isolated mitochondria and offers a means of analyzing yeast mutants defective in mitochondrial protein import. The in vitro import of an artificial precursor protein into yeast mitochondria in the presence of its substrate analog was analyzed subsequent to synthesis in either a yeast or rabbit reticulocyte cell-free translation reaction. Results suggest that a component(s) present in the yeast cytosolic extract may interact with the precursor protein.     

Role of heat shock cognate 70 protein in import of ornithine transcarbamylase precursor into mammalian mitochondria.

The roles of the 70-kDa cytosolic heat shock protein (hsp70) in import of precursor proteins into the mitochondria were postulated to be related to (i) unfolding of precursor proteins in the cytosol, (ii) maintenance of the import-competent state, and (iii) unfolding and transport of precursor proteins through contact sites, in cooperation with matrix hsp70. We examined roles of cytosolic hsp70 family members in import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria, using an in vitro import system and antibodies against hsp70. Immunoblot analysis using an hsc70 (70-kDa heat shock cognate protein)-specific monoclonal antibody and a polyclonal antibody that reacts with both hsc70 and hsp70 showed that hsc70 is the only or major form of hsp70 family members in the rabbit reticulocyte lysate. The hsc70 antibody did not inhibit pOTC import when added prior to import assay. However, when pOTC was synthesized in the presence of the antibody and then subjected to import assay, pOTC import was markedly decreased. pOTC import was also decreased when the precursor was synthesized in the lysate depleted for hsc70 by treatment with hsc70 antibody-conjugated Sepharose. This reduction was almost completely restored by readdition of purified mouse hsc70 during pOTC synthesis. The readdition of hsc70 after pOTC synthesis and only during the import assay was not effective. Thus, once import competence of pOTC was lost, hsc70 was ineffective for restoration. Newly synthesized pOTC lost import competence in the absence of hsc70 somewhat more rapidly than in its presence. These results indicate that hsc70 is required during pOTC synthesis and not during import into the mitochondria. hsc70 presumably binds to pOTC polypeptide and maintains it in an import-competent form.     

Inhibition of the import of mitochondrial proteins by RNase

RNase treatment of a cell-free translation system prevents transport of mitochondrial precursor proteins from that system into isolated yeast mitochondria. This inhibition depends on the presence of ribosomes in the reticulocyte lysate; if they are cleared by centrifugation, RNase treatment does not specifically inhibit protein uptake by mitochondria. Since protein import can occur in the absence of polyribosomes, RNase treatment does not degrade a structure essential for this process. Rather, the inhibition may be an effect of degraded ribosomes.