Translation Products in Isolated Mitochondria from Neurospora crassa: Partial Assembly of Cytochrome Oxidase Components
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The transport of cytoplasmically synthesized mitochondrial proteins was investigated in whole cells of Neurospora crassa, using dual labelling and immunological techniques.In pulse and pulse-chase labelling experiments the mitochondrial proteins accumulate label. The appearance of label in mitochondrial protein shows a lag relative to total cellular protein, ribosomal, microsomal and cytosolic proteins.The delayed appearance of label was also found in immunoprecipitated mitochondrial matrix proteins, mitochondrial ribosomal proteins, mitochondrial carboxyatractyloside-binding protein and cytochrome c. Individual mitochondrial proteins exhibit different labelling kinetics.Cycloheximide inhibition of translation does not prevent import of proteins into the mitochondria. Mitochondria1 matrix proteins labelled in pulse and pulse-chase experiments can first be detected in the cytosol fraction and subsequently in the mitochondria. The cytosol matrix proteins and those in the mitochondria show a precursor-product type relationship.The results suggest that newly synthesized mitochondrial proteins exist in an extra-mitochondria1 pool from which they are imported into the mitochondria.The majority of mitochondrial proteins are coded for by nuclear genes and are synthesi~ed on cytoplasmic ribosomes. It has been estimated that some 85 -95 % of the total mitochondrial protein originates from cytoplasmic protein synthesis [l]. These proteins must subsequently be transported to their functional locations in the mitochondrion. A similar situation exists in the formation of chloroplasts in plant cells where the majority of the chloroplast proteins originate in the cytoplasm [2].Despite the obvious importance of the transport process little is known about the selectivity which allows only mitochondrial proteins access to mitochondria and about the transport mechanism. In recent years a mechanism was proposed which integrates synthesis and transport into a simple unified mechanism [ 3 -61. According to this attractive hypothesis, mitochondrial proteins are translated by cytoplasmic ribosomes which are bound to the outer membrane of the mitochondria. Nascent polypeptides are discharged in a vectorial manner into the matrix, or membranes, in a manner analogous to the mechanism proposed for protein transport in exocrine cells [7]. The evidence for the general existence of such a mechanism is equivocal and many questions in relation to how such a system could function remain unanswered.We have studied the kinetics of synthesis of mitochondrial proteins and their subsequent appearance in the mitochondria in intact cells of Neurospora cru,ssu. The results of our studies suggest that extramitochondrial pools of mitochondrial proteins exist. It is furthermore shown that newly synthesized mitochondrial proteins can be detected outside the mitochondrial fraction. Our data are not readily compatible with a mechanism in which synthesis and transport are functionally linked. The existence of cytoplasmic ribosomes bound to outer membranes of mi...
The transport of cytoplasmically synthesized mitochondrial proteins was investigated in whole cells of Neurospora crassa, using dual labelling and immunological techniques.In pulse and pulse-chase labelling experiments the mitochondrial proteins accumulate label. The appearance of label in mitochondrial protein shows a lag relative to total cellular protein, ribosomal, microsomal and cytosolic proteins.The delayed appearance of label was also found in immunoprecipitated mitochondrial matrix proteins, mitochondrial ribosomal proteins, mitochondrial carboxyatractyloside-binding protein and cytochrome c. Individual mitochondrial proteins exhibit different labelling kinetics.Cycloheximide inhibition of translation does not prevent import of proteins into the mitochondria. Mitochondria1 matrix proteins labelled in pulse and pulse-chase experiments can first be detected in the cytosol fraction and subsequently in the mitochondria. The cytosol matrix proteins and those in the mitochondria show a precursor-product type relationship.The results suggest that newly synthesized mitochondrial proteins exist in an extra-mitochondria1 pool from which they are imported into the mitochondria.The majority of mitochondrial proteins are coded for by nuclear genes and are synthesi~ed on cytoplasmic ribosomes. It has been estimated that some 85 -95 % of the total mitochondrial protein originates from cytoplasmic protein synthesis [l]. These proteins must subsequently be transported to their functional locations in the mitochondrion. A similar situation exists in the formation of chloroplasts in plant cells where the majority of the chloroplast proteins originate in the cytoplasm [2].Despite the obvious importance of the transport process little is known about the selectivity which allows only mitochondrial proteins access to mitochondria and about the transport mechanism. In recent years a mechanism was proposed which integrates synthesis and transport into a simple unified mechanism [ 3 -61. According to this attractive hypothesis, mitochondrial proteins are translated by cytoplasmic ribosomes which are bound to the outer membrane of the mitochondria. Nascent polypeptides are discharged in a vectorial manner into the matrix, or membranes, in a manner analogous to the mechanism proposed for protein transport in exocrine cells [7]. The evidence for the general existence of such a mechanism is equivocal and many questions in relation to how such a system could function remain unanswered.We have studied the kinetics of synthesis of mitochondrial proteins and their subsequent appearance in the mitochondria in intact cells of Neurospora cru,ssu. The results of our studies suggest that extramitochondrial pools of mitochondrial proteins exist. It is furthermore shown that newly synthesized mitochondrial proteins can be detected outside the mitochondrial fraction. Our data are not readily compatible with a mechanism in which synthesis and transport are functionally linked. The existence of cytoplasmic ribosomes bound to outer membranes of mi...
Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in a Cell Free System from Neurospora crassa
Synthesis and transport of mitochondrial proteins were followed in a cell-free homogenate of Neurospora crussa in which mitochondrial translation was inhibited. Proteins synthesized on cytoplasmic ribosomes are transferred into the mitochondrial fraction. The relative amounts of proteins which are transferred in vitro are comparable to those transferred in whole cells.Cycloheximide and puromycin inhibit the synthesis of mitochondrial proteins but not their transfer into mitochondria.The transfer of immunoprecipitablc mitochondrial proteins was demonstrated for matrix proteins, carboxyatractyloside-binding protein and cytochrome c.Import of proteins into mitochondria exhibits a degree of specificity. The transport mechanism differentiales between newly synthesized proteins and preexistent mitochondrial proteins, at least in the case of matrix proteins.In the cell-free homogenate membrane-bound ribosonies are more active in the synthesis of mitochondrial proteins than are free ribosomes. The finished translation products appear to be released from the membrane-bound ribosomes into the cytosol rather than into the membrane vesicles.The results suggest that the transport of cytoplasniically synthesized mitochondrial proteins is essentially independent of cytoplasmic translation ; that cytoplasmically synthesized mitochondrial proteins exist in an extramitochondrial pool prior to import; that the site of this pool is the cytosol for at least some of the mitochondrial proteins; and that the precursors in the extramitochondrial pool differ in structure or conformation from the functional proteins in the mitochondria.In the preceding paper we studied the transport of cytoplasmically synthesized proteins into the mitochondria in intact cells of Neurosporu crassu [l]. la whole cells, the processes of synthesis and transport are not easily separated. Furthermore, post-labelling fractionation artifacts may obscure the intracellular location of mitochondrial proteins which are on their path from the site of synthesis to the site of function.In considering systems in vitro a number of possibilities exist, ranging Crom cell-free homogenates to reconstituted systems involving isolated and purified protein and organelle components. In the present study we have investigated synthesis and transport in a cell-free homogenate. We. have employed this system in an attempt lo decide between two mechanisms currently proposed for the transport of mitochondria] proteins. The first mechanism is based on observations with whole IC'emrospora cells [l]. This mechanism proposes the existence of extramitochondrial pools of mitochondrial proteins from which they are imported into the mitochondria. The second mechanism is that proposed by Butow and his colleagues [3 -61. This latter mechanism proposes a special class of cytoplasmic ribosomes which are attached to the mitochondria at specific sites. The nascent polypeptides from these ribosomes are discharged in a vectorial manner into the mitochondria.The results we present here support our earli...
A simple method for the isolation of rat liver cells is described. The cells are shown, by an isotope dilution method, to maintain a constant rate of protein synthesis for 8 h of incubation. Antibodies to purified rat liver cytochrome oxidase were raised in rabbits and used to investigate the labeling of cytochrome oxidase in isolated rat liver cells and in vivo. The data demonstrate the occurrence of a precursor of the subunits of cytochrome oxidase that are synthesized in the cytoplasm.1. Dodecylsulfate gel electrophoresis of the immunoprecipitates from isolated rat liver cells that had been labeled with [35S]methionine for 1 h showed a single radioactive peak with a molecular weight of 50000.2. Judged by the effects of cycloheximide and chloramphenicol the labeled protein is synthesized on cytoplasmic ribosomes.3. After labeling for 1 h in vivo with [3H]leucine the labeled protein appears to be exclusively associated with the hepatic niicrosomal fraction.4. Ouchterlony double-diffusion analysis demonstrated immunological relationship between the precipitates from microsomes and cytochrome oxidase. In addition to the precipitates derived from mitochondria and microsomes immunoprecipitates were also obtained from the cytosol in comparable amounts ; these again were immunologically related.The occurrence of large amounts of precursor(s) (or degradation products) of cytochrome oxidase in rat liver fractions is interpreted in terms of a regulatory pool for amino acid homeostasis in the organism.Cytochrome oxidase, the terminal oxygen acceptor of the respiratory chain in the inner mitochondrial membrane, represents a hydrophobic enzyme complex that is composed of seven protein subunits, two heme a groups, two copper atoms and tightly bound lipids (for reviews see [l-31). The biosynthesis of the enzyme is a complex process involving the assembly of four small subunits that are coded for in the nucleus and synthesized on cytoplasmic ribosomes, and three larger subunits that are coded for and synthesized within the mitochondria (for reviews see . Although these results are mainly based on studies with yeast similar data on subunit composition [9, of the cytoplasmically synthesized subunits and on the mechanism of assembling the enzyme complex. Poyton et al. described the extramitochondrial occurrence of a precursor protein for the four small subunits of yeast cytochrome oxidase which stimulates the synthesis of the three large subunits in mitochondria and which is immunoprecipitated from a postpolysomal supernatant fraction by antibodies to the holoenzyme 129,301. This protein has a molecular weight of 55000 and is thought to contain a small peptide in addition to the sequences of the four cytoplasmically synthesized subunits of cytochrome oxidase. Rucker et al. also presented evidence for the occurrence of precursor subunits VI and VII in the postribosomal cytoplasmic supernatant fraction of Neurospora crassa cells [31]. An intermediate in the assembly of mitochondria1 and cytoplasmically synthesized subunits of cy...
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