Isolation of a subfraction of rough endoplasmic reticulum closely associated with mitochondria : Evidence for its role in cytochrome P450 synthesis

A subfraction of rough endoplasmic reticulum (RER) structurally associated with mitochondria (mito-RER complexes) was isolated from crude nuclear fractions of rat liver homogenate. When apocytochrome P450 synthesis (which presumably occurs in RER) and mitochondrial heme synthesis was dissociated by concomitant treatment of rats with phenobarbital and cobaltous chloride, apocytochrome P450 accumulated predominantly in mito-RER complexes. These data suggest that cytochrome P450 synthesis requires structural interaction of mitochondria and RER.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : II. Glucose-6-Phosphatase in Rough Microsomes

Electron microscope cytochemical localization of glucose-6-phosphatase in the developing hepatocytes of fetal and newborn rats indicates that the enzyme appears simultaneously in all the rough endoplasmic reticulum of a cell, although asynchronously within the hepatocyte population as a whole. To confirm that the pattern of cytochemical deposits reflects the actual distribution of enzyme sites, a method to subfractionate rough endoplasmic reticulum was developed. The procedure is based on the retention of the cytochemical reaction product (precipitated lead phosphate) within freshly prepared rough microsomes reacted in vitro with glucose-6-phosphate and lead ions. Lead phosphate increases the density of the microsomes which have glucose-6-phosphatase activity and thereby makes possible their separation from microsomes lacking the enzyme; separation is obtained by isopycnic centrifugation on a two-step density gradient. The procedure was applied to rough microsomes isolated from rats at several stages during hepatocyte differentiation and the results obtained agree with those given by cytochemical studies in situ. Before birth, when only some of the cells react positively for glucose-6-phosphatase, only a commensurate proportion of the rough microsome fraction can be rendered dense by the enzyme reaction. At the time of birth and in the adult, when all cells react positively, practically all microsomes acquire deposit and become dense after reaction. Thus, the results of the microsome subfractionation confirm the cytochemical findings; the enzyme is evenly distributed throughout all the endoplasmic reticulum of a cell and there is no regional differentiation within the rough endoplasmic reticulum with respect to glucose-6-phosphatase. These findings suggest that new components are inserted molecule-by-molecule into a pre-existing structural framework. The membranes are thus mosaics of old and new molecules and do not contain large regions of entirely "new" membrane in which all of the components are newly synthesized or newly assembled.     

Chemical and Biochemical Changes in Subcellular Fractions of Guinea Pig Liver During Infection with Coxiella burneti

Livers of uninfected guinea pigs and of guinea pigs infected with Coxiella burneti were fractionated into smooth endoplasmic reticulum, rough endoplasmic reticulum (RER), pellet, and cell sap fractions. The ribonucleic acid (RNA) and protein of each fraction were determined, and the phosphorylase, glucose-6-phosphatase, and glucosyl transferase (glycogen synthetase) activities of each fraction were measured. Decreased RNA, protein, and enzyme activities were found in the RER and pellet fractions of infected livers, with the greatest differences in the RER. The evidence indicates a solubilization of the phosphorylase and synthetase, with the enzymes moving from the RER and glycogen-containing pellet fraction to the cell sap. The data suggest the RER as a target during Q fever.     

Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver.

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.     

The characteristics of liver glucose-6-phosphatase in the envelope of isolated nuclei and microsomes are identical

We have compared the characteristics of glucose-6-phosphatase (EC 3.1.3.9) in the envelope of purified nuclei and microsomes from rat liver. The latency of mannose-6-P hydrolysis, permeability to EDTA, and susceptibility of the enzyme to protease-mediated inactivation all indicated that the permeability barrier defined by the envelope in situ is significantly disrupted in isolated nuclei (i.e. in vitro). Latency of mannose-6-P hydrolysis was demonstrated to provide a quantitative measure of the degree of nuclear membrane disruption. Electron micrographs confirmed the existence of substantial regions of the envelope in vitro where the permeability barrier to EDTA was intact (i.e. an "intact component"). The kinetics of glucose-6-phosphatase catalyzed by the intact component was obtained by subtracting the contribution of enzyme in disrupted regions from the total enzymic activity of untreated nuclei. The characteristics of glucose-6-phosphatase in intact and fully disrupted membranes of nuclei were indistinguishable from microsomes with respect to (a) the kinetics of glucose-6-P hydrolysis, (b) the effects of incubations with mannose-6-P, N-ethylmaleimide, and protease from Bacillus amyloliquefaciens, (c) the extremely high latency of carbamyl phosphate:glucose phosphotransferase activity, and (d) both the patterns of response of activity and the change in latency of glucose-6-phosphatase induced by fasting, experimental diabetes, and cortisol injection. Our results show clearly that apparent differences in the glucose-6-phosphatase activity of untreated preparations of nuclei and microsomes are simply expressions of significant differences in the degree of intactness of their respective permeability barriers. Since flattened cisternae, characteristic of the rough endoplasmic reticulum in situ, are preserved in intact regions of the envelope of isolated nuclei, the present findings constitute the most direct and definitive evidence to date that the properties of glucose-6-phosphatase in the endoplasmic reticulum in situ are faithfully reproduced with intact microsomes.     

The physical association between rat liver mitochondria and rough endoplasmic reticulum : I. Isolation, electron microscopic examination and sedimentation equilibrium centrifugation analyses of rough endoplasmic reticulum-mitochondrial complexes

Rough endoplasmic reticulum-mitochondrial (RER-MT) complexes have been isolated from rat liver homogenates by rate zonal Centrifugation using a reorienting zonal rotor. Electron microscopic examination of the isolated complexes reveals a close association between rough endoplasmic reticulum (RER) and mitochondria. The associated RER appears as bilamellar sheets as it does in intact liver tissue, not as microsomal vesicles. When the complexes are subjected to sedimentation equilibrium Centrifugation, the marker enzymes for mitochondria and RER coband at an equilibrium density of 1.190. Electron microscopic analysis of the complexes after sedimentation equilibrium Centrifugation again reveals a close association between RER and mitochondria. Treatment of the complexes with 500 mM KCl or 500 mM KCl plus 20 mM EDTA resulted in a shift in the equilibrium density of the complexes to 1.180 and 1.176, respectively. Concomitant with the density shift was a release of A₂₆₀ units to the top of the gradient. After incubating KCl-EDTA stripped complexes with cytoplasmic ribosomes and ribosomal subunits, the complexes band at the same equilibrium density, 1,190, as do untreated complexes. In order to completely remove the associated RER it is necessary to treat the complexes with digitonin at a concentration of 0.13 mg digitonin/mg protein. Our data suggest that a fraction of the total cellular RER is physically associated with rat liver mitochondria.     

Properties of a rat liver smooth microsomal subfraction not aggregated by Mg2+

A smooth microsomal fraction (smooth II microsomes) was earlier isolated and characterized in a number of investigations. Using a three-layer discontinuous sucrose gradient containing Mg²⁺ this fraction was divided into two subfractions (IIa and IIb) by a single centrifugation. The smooth IIa fraction proved to be a purified smooth microsomal fraction of specific composition. It contains high amounts of cytochromes $\text{b}{}_5$ and P-450, low activities of other electron transport enzymes and glucose-6-phosphatase, and no UDP-glucuronic acid transferase. No membrane or enzyme synthesis is induced in this subfraction by treatment with phenobarbital or methylcholanthrene. It appears that the membranes of smooth IIa microsomes derive from the smooth endoplasmic reticulum and are devoted to specific functions.     

Phosphatase cytochemistry with cerium as trapping agent. Verification of acid phosphatase and glucose-6-phosphatase reactive sites

Lead is prevalently replaced by cerium as trapping agent in phosphatase cytochemistry to prevent non-specific precipitation. Recently, substrate specific but artefactual lead precipitates have been described in the nuclear envelope (NE) and rough endoplasmic reticulum (RER) due to a local matrix effect. In the present study a verification was carried out of the localization of acid phosphatase and glucose-6-phosphatase in the NE and RER of rat peritoneal macrophages and hepatocytes respectively with cerium. It appeared that precipitates of cerium phosphate in NE and RER of peritoneal macrophages do not represent sites of acid phosphatase activity but are due to the matrix effect. However, in rat hepatocytes these organelles demonstrate true reactive sites for glucose-6-phosphatase.     

Phosphatase cytochemistry with cerium as trapping agent

Summary Lead is prevalently replaced by cerium as trapping agent in phosphatase cytochemistry to prevent nonspecific precipitation. Recently, substrate specific but artefactual lcad precipitates have been described in the nuclear envelope (NE) and rough endoplasmic reticulum (RER) due to a local matrix effect. In the present study a verification was carried out of the localization of acid phosphatase and glucose-6-phosphatase in the NE and RER of rat peritoneal macrophages and hepatocytes respectively with cerium. It appeared that precipitates of cerium phosphate in NE and RER of peritoneal macrophages do not represent sites of acid phosphatase activity but are due to the matrix effect. However, in rat hepatocytes these organelles demonstrate true reactive sites for glucose-6-phosphatase.In honour of Professor van Duijn     

Cytoplasmic changes in level and distribution of glucose-6-phosphatase activities from rat liver during diethylnitrosamine-induced carcinogenesis.

Glucose-6-phosphatase (EC 3.1.3.9) activities were determined in isolated microsomes, cytoplasmic smooth and rough membranes, ribosomes and free cytosol from rat liver undergoing carcinogenesis by diethylnitrosamine (DENA) and compared with cytoplasmic fractions isolated in parallel from healthy animals from the same age.With continuous administration of a low dose of DENA (2.6 mg/kg rat per day for 20 weeks in the drinking water) livers of carcinogen treated rats became heavier than the control livers but the body weight decreased. About 70% of total glucose-6-phosphatase activity could be detected in the microsomal fraction. While there was no significant difference in this activity in both animal groups up to the 4th week, glucose-6-phosphatase of cancerous liver showed a distinct decrease of activity compared with normal liver.During cancer induction this enzyme became more soluble, confirmed by the observation that it was detached from firmer structures of cytoplasm as rough membranes and polysomes and translocated to smooth membranes and the soluble cytoplasmic fraction successively. The corresponding increase in glucose-6-phosphatase activity in the 105 000 g supernatant appears to be due to the loss of enzyme activity in a distinct cytoplasmic membrane fraction. These data strongly suggest that in parallel with alteration of cytoplasmic membrane structures during carcinogen feeding glucose-6-phosphatase is detached from heavier components of the cytoplasm while total activity decreased. Possible mechanisms of these findings are discussed.     

ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER : II. Enzymatic Characterization and Comparison with Other Cell Fractions

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20–30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2–3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.     

Reversibility of lysosomal and glucose 6-phosphatase changes produced in the rat liver by dimethylnitrosamine

The present investigation was undertaken to discover whether repeated doses of dimethylnitrosamine (DMNA) could produce a cumulative toxic effect on the rat liver. For this purpose doses were selected at a level just too low to produce cytopathological changes, as indicated by depression of glucose-6-phosphatase and induction of autophagic vacuoles (AV) in hepatocytes, when given once only. Single subcutaneous injections of 10 or 3 mg/kg induced these cytopathological changes in the centrilobular (CLB) hepatic cells but when the dose was reduced to l mg/kg no such changes were seen.

After daily administration of l mg/kg for 4 or 8 weeks we observed both glucose-6-phosphatase depression and autophagy, and in addition there was marked hypertrophy of the rough endoplasmic reticulum, nucleolar microsegregation and the appearance of distorted, often ring-shaped mitochondria with shortened cristae. Kupffer cells exhibited a marked increase in lysosomal activity. With the exception of mitochondrial changes and Kupffer cell activity this same picture was observed, although in milder form, when the dose administered was 0.3 or 0,1 mg/kg daily for the same period. When treatment was continued for 12 weeks, however, the only differences from control rats were the presence of hypertrophied rough endoplasmic reticulum (RER) at all three dose levels, nucleolar microsegregation at the upper two dose levels, and pronounced Kupffer cell activity at the top dose. These findings indicate that cumulative cytopathologic effects occur only up to 8 weeks at the dose levels studied but hypertrophy of RER and increased Kupffer cell activity persist up to 12 weeks.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : I. Glucose-6-Phosphatase Distribution In Situ

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.     

A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum.

We sought a rapid and non-ultracentrifugal method of recovering large amounts of highly pure rough endoplasmic reticulum (RER) membranes from livers. By substantially modifying a 20-year-old calcium precipitation technique, we obtained a RER fraction from rat liver and established its high degree of purity by quantitating classic membrane markers for different subcellular organelles. This RER fraction is highly enriched in four known proteins (or enzyme activities) required for lipoprotein assembly: apolipoprotein B, microsomal triglyceride transfer protein, acyl CoA:diacylglycerol acyltransferase, and acyl CoA:cholesterol acyltransferase, when compared to two classical RER markers, RNA and glucose-6-phosphatase. From one 10-12 g rat liver, we recover ten to twelve RER pellets of 1.5-1.6 cm in diameter containing approximately 110-125 mg of total protein, about half of which is sodium carbonate-releasable. By electron microscopy, these large RER pellets from rat livers are homogeneously comprised largely of non-vesiculated short strips of ribosome-rich membranes. This novel technique for isolating RER membranes from liver may provide a useful tool for future studies on the assembly of apolipoprotein B-containing lipoproteins as well as for research focused on mechanisms of secretory and membrane protein translation, translocation, and folding.     

Effect of diabetes on the rat hepatic glucose-6-phosphatase system in endoplasmic reticulum subfractions

Diabetes-induced alterations in the activities of the components of the glucose-6-phosphatase system (i.e., the enzyme, the glucose-6-P translocase (T(1)), and the phosphate translocase (T(2)) were examined in smooth and rough subfractions of hepatic endoplasmic reticulum from streptozotocin-injected rats. A significant effect of diabetes on the maximal velocity of glucose-6-P hydrolysis by the enzyme was present in both endoplasmic reticulum subfractions (3.1-fold increase in rough endoplasmic reticulum; 3.8-fold increase in smooth endoplasmic reticulum). Based on latency values, diabetes did not result in a proportional increase in capacity of T(1) or T(2). In contrast to the control condition, the relationship between transport capacity and hydrolytic capacity was not significantly different in the two subfractions from diabetic animals. Elucidation of the effects of diabetes on the components of the glucose-6-phosphatase system associated with smooth and rough endoplasmic reticulum membranes enhances our understanding of the hepatic contribution to diabetic hyperglycemia.     

Phenobarbital-induced alterations in the activities of the transport and hydrolytic components of the glucose-6-phosphatase system in smooth and rough subfractions of the rat hepatic endoplasmic reticulum

We have examined the influence of the phenobarbital-induced proliferation of the hepatic endoplasmic reticulum (ER) on the activities of the components of the glucose-6-phosphatase system, i.e., the enzyme, the glucose-6-P translocase (T1), and the phosphate translocase (T2). Young male rats were injected ip twice daily for 4 days with 4 mg/100 g body wt of phenobarbital (PB) or an equivalent volume of saline solution. On the fifth day, the rats were killed and smooth (SER) and rough (RER) fractions of the ER were isolated from liver homogenates. Kinetic constants for glucose-6-P hydrolysis by the system and enzyme were determined and used to calculate the kinetic constants for glucose-6-P transport. T2 activity was approximated by assaying the pyrophosphatase activity at pH 6.0 in intact microsomes. Three times more SER protein was recovered from livers of PB-treated rats. PB-treatment did not alter total liver enzyme activity, but total liver T1 activity was decreased to 59% of the control value. Maximal specific activities of the system, enzyme and T1 were all reduced by PB treatment to 44% of control values in the RER and to 68% of control values in the SER. PB treatment reduced the apparent activity of T2 in RER and SER to 35 and 49% of the respective control values. In the SER from both groups of rats, T1 activity or apparent T2 activity divided by enzyme activity was about 55% of the corresponding ratio in the RER. Our analysis of these data suggests that the lower activities of T1 and T2 in the smooth ER are the results of suppression by some intrinsic component localized in the smooth membrane. Accordingly, the reduction in total liver T1 activity and, therefore, system activity in PB-treated rats reflects the redistribution of the glucose-6-P translocase from the RER to the more abundant SER membrane where it is less active. The possibility is discussed that a higher cholesterol content within the SER membrane is responsible for the lower transport activities.     

Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjection of rat liver microsomes into Xenopus oocytes

Fragments of rough and smooth endoplasmic reticulum purified from rat liver were injected into Xenopus oocyte cytoplasm. Light and electron microscopy, cytochemistry, immunocytochemistry, and enzyme assay were employed to determine the fate of heterologous membranes in the host cytoplasm. The in vivo-incubated microsomes disappeared in a time-dependent manner. Within 3 hr, rough microsomes were replaced by flattened ER cisternae and smooth microsomes were replaced by a network of anastomosing tubules. Polyclonal antibodies against rat liver microsomes and protein A-gold complexes were applied to glycol methacrylate sections of microinjected oocytes. Specific labeling was observed over discrete rough and smooth ER cisternae 3 hr after microinjection. Endogenous ER was not labeled by this technique, and label was not observed when sections were treated with pre-immune antibodies. Diaminobenzidene cytochemistry of microinjected rat lacrimal gland microsomes revealed enzyme activity in heterologous microsomes after 3 hr of in vivo incubation. Control injected microsomes (inactivated by heat denaturation) became associated with autophagic vacuoles, coincident with changes in lysosomal activity. Freshly isolated un-denatured microsomes did not provoke changes in lysosomal activity, and glucose-6-phosphatase activity associated with microinjected membranes could be detected 21 hr after in vivo incubation. Since rat liver microsomes reconstitute after in vivo incubation into cytoplasmic structures resembling those from which they were derived, we conclude that the microinjected membrane fragments act as templates for their own three-dimensional organization.     

Exchange of phospholipids between mitochondria and rough or smooth microsomes in vitro.

Phospholipids in mitochondria can be exchanged with those in two microsomal fractions from rough endoplasmic reticulum (rough microsomes) and smooth endoplasmic reticulum (smooth microsomes) in vitro in the presence of cell supernatant. The amounts of phospholipids transferred from each submicrosomal fraction to nitochondria were slightly different. The compositions of the phospholipids transferred to mitochondria from both microsomal fractions were the same, though these two fractions actually had different phospholipid compositions.     

The localization of cholinephosphotransferase in the outer membrane of guinea-pig lung mitochondria

The activity of cholinephosphotransferase was measured in the subcellular fractions of guinea-pig lung. The specific activity of the enzyme was highest in a fraction, intermediate in density between mitochondria and microsomes. Similar subcellular distribution patterns were observed for both cholinephosphotransferase and rotenone-insensitive NADH-cytochrome c reductase, an enzyme associated with the outer membrane of mitochondria and endoplasmic reticulum, suggesting that cholinephosphotransferase may be localized in both of these organelles. The distribution of cholinephosphotransferase activity in the subfractions of mitochondria and the intermediate fractions recovered by linear density gradient paralleled that of the mitochondrial outer membrane marker enzyme, monoamine oxidase. RNA content of a subfraction enriched in cholinephosphotransferase and monoamine oxidase was not typical to that of either rough or smooth endoplasmic reticulum. The results of this study suggest that in guinea-pig lung, cholinephosphotransferase is localized in both the outer membrane of mitochondria, and the endoplasmic reticulum.     

Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.