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The use of puromycin in vivo and EDTA in vitro , both separately and together, has made it possible to eliminate peptidyl-t-RNA and divalent metal cations, both separately and together, as agents responsible for attaching ribosomes to the endoplasmic reticulum of rat liver. Quantitative support has been obtained for the concept that ribosomes are bound to the ER membrane by their larger subunit, with their smaller subunit attached to the larger by magnesium ions. This has been found for rats, mice and guinea pigs.

Isolated mitochondria cannot synthesize their own phospholipids, there is only an exchange between exogenous and mitochondrial phospholipid fatty acids. In vitro, the endoplasmic reticulum phospholipids exchange with the phospholipids of the mitochondrial outer and inner membranes. Exchange of the endoplasmic reticulum phospholipids with those of the inner membrane is the same when the incubation is carried out with whole mitochondria or with mitochondria devoid of outer membranes.

Several types of intimate association are shown between endoplasmic reticulum or endoplasmic reticulum-like membranes and the outer membranes of mitochondria in fungal hyphae. These include close physical association, contact, thread-like continuity, and direct luminal continuity. Membranes are smooth surfaced in the immediate region of association or continuity, and some have ribosomes at other sites along their surfaces. The continuities represent sites of membrane interaction which may facilitate exchange between adjacent membrane components. Additional continuities are shown between mitochondria and other endomembrane components. Observations are discussed in relation to the body of information linking mitochondria and endoplasmic reticulum in a variety of eukaryotic cells.

Two isoantigenic variants of an A/Sn X A.SW lymphoma (LNSF) were separated into the following subcellular fractions: soluble material, total subcellular particulates, “microsomes”, plasma membrane and endoplasmic reticulm. The distribution in these fractions of 6 H-2 specificities was studied using

Summary Following an interval of incorporation of radioactive δ-aminolevulinic acid, the pattern of labeling of the multiple forms of cytochrome c of rat liver tissue was determined. The results obtained indicate that the product of the synthetic activity of the endoplasmic reticulum must be a minor, nonfunctional form (Form IV) and that it is this entity which is transferred to the mitochondrion from the endoplasmic reticulum.

Golgi apparatus from rat liver were higher or equal to endoplasmic reticulum in phosphatidylethanolamine and phosphatidylinositol but were intermediate between endoplasmic reticulum and plasma membrane with respect to levels of phosphatidylcholine, phosphatidylserine, sphingomyelin, and sterols. Endoplasmic reticulum was highest in phosphatidylcholine and lowest in phosphatidylserine, sterols, and sphingomyelin. Polyacrylamide gel disc electrophoretic patterns of structural and total protein fractions showed unique band patterns for each of the above three fractions as well as mitochondria. Patterns for total protein of Golgi apparatus and endoplasmic reticulum were similar, but endoplasmic reticulum had three additional bands absent from Golgi apparatus. Plasma membrane revealed the simplest pattern with one major band. A corresponding band was present in patterns from Golgi apparatus, endoplasmic reticulum, and mitochondria. Golgi apparatus were found to be compositionally distinct from other fractions examined and intermediate between endoplasmic reticulum and plasma membrane. The results are compatible with the concept of partial derivation of Golgi apparatus membranes from endoplasmic reticulum as well as the hypothesis that the Golgi apparatus functions in transformation of membranes from endoplasmic reticulum-like to plasma membrane-like.

The effects of glycolysis of a rat-kidney microsomal membrane preparation have been studied. The membrane preparation stimulates glycolytic activity more than 50%. The site of this stimulation is shown to be the 3-phosphoglycerate kinase reaction. Evidence is presented that the stimulation is due to ATPase activity of the membrane interacting with ATP associated with the kinase enzyme. The membrane preparation is shown to oxidize DPNH generated by glycolysis. The observed interaction between enzymes glycolysis and the membrane is interpreted as a channeling of metabolic energy into the membrane for active transport and demonstrates one mechanism whereby active transport stimulates a metabolic pathway.

A survey of a large number of different cell types has indicated the presence of a network of membrane-bound cavities (the endoplasmic reticulum) in the cytoplasm of all cell types examined, with the exception of the mature erythrocyte. In its simplest form, encountered in seminal epithelia and in leucocytes, the reticulum consists mainly of interconnected strings of vesicles and appears to be randomly disposed in three dimensions. Local differentiations occur within the endoplasmic reticulum of all the cell types studied. The membrane limiting the cavities of the endoplasmic reticulum appears to be continuous with the cell membrane and the nuclear membranes.

A method for the fine structural localization of acyltransferases catalyzing the transfer of acyl groups from palmityl-coenzyme A (CoA) to α-glycerophosphate based on the formation of a heavy metal precipitate at the site of CoA release is described. In this method CoA released through the action of the enzyme is oxidized by potassium ferricyanide, which is reduced to potassium ferrocyanide and precipitates in the presence of cupric ions. Acyltransferase activity was found to survive both fixation in glutaraldehyde and incubation in the presence of the capture reagent system used for cytochemistry. Reaction product marking the enzyme activity was located within the cisternae of the rough endoplasmic reticulum and to lesser, but significant extent, within the membranous envelope of the smooth endoplasmic reticulum. Reaction product was not associated to any significant extent with other membranous organelles. The significance of these observations in terms of the role of acyltransferases in liver function is discussed.

1. 1. Rough endoplasmic reticulum membrane from rat liver was stripped of its ribosomes and was then reacted with liver polyribosomes. Polysomes derived from whole-cell preparations and polysomes free in the cytoplasm were equally effective in providing reconstituted rough endoplasmic reticulum. 2. 2. Reconstituted rough endoplasmic reticulum was tested in vitro for capacity to incorporate amino acids from aminoacyl-tRNA into peptide. The presence of the membrane resulted in a requirement for sulfhydryl compounds not needed by the same polysomes incubated in the absence of membrane. This modification imposed by the presence of the membrane occurred whether the polysomes used for reconstitution were representative of the whole cell population or were polysomes originally free in the cytoplasm. However, polysomes made by adding polyuridylic acid to ribosome subunits did not show this sulfhydryl dependence when used for reconstitution of rough endoplasmic reticulum. This may have been related to the fact that most of these polyuridylic acid complexes were monomers. 3. 3. It is concluded that peptide bond formation by the reconstituted system resembles that of the original rough endoplasmic reticulum freshly prepared from the liver cell in having a sulfhydryl requirement due to the presence of the membrane.

Acid hydrolases are not exclusively lysosomal enzymes as indicated by the specific case of β-glucuronidase. This enzyme is a membrane protein found in the endoplasmic reticulum and in the lysosomes of the cell.

A subfraction of membrane protein has been isolated from the microsomal fraction of rat liver. This material has blocked N-terminal amino groups and a distribution of C-terminal amino acids, indicating that the membrane protein preparation is composed of distinct chemical species. However, it has been demonstrated that the species present comprise a reversibly associating system. Furthermore, the characteristics of this system are such that the species involved are probably related to one another. Additional information shows that the protein is associated with nucleotide-like material in a ratio of 1 mole of nucleotide per 10 000 g of protein. The preparation has also been characterized with respect to relative contents of cysteic acid, methionine sulfone, proline and glutamic acid. The ratios obtained from the experimental values may be used to identify the protein subfraction and distinguish the preparation from membrane protein obtained from other sources.

The semipermeability of microsomal membranes has certain important functional implications, mainly since the accessibility of a number of substrates to the membrane-bound enzymes is shown to be a limiting factor [l-3] . Changes in membrane permeability by external or internal agents may be decisive for regulating the activity of various enzymic systems [4,5] . Permeability properties also determine the size and density of the particle, which are the most important factors in the subfractionation of microsomes [6] . For the above reasons, the permeability of microsomal membranes was investigated in vitro, and experiments were performed to see whether the reaction products of one of the main microsomal enzymic systems, the hydroxylation complex, are actively transferred through the membrane during in vitro conditions.

An electron microscope study of developing mouse oocytes has revealed a close morphological relationship between mitochondria and endoplasmic reticulum. In many instances, it was noted that the outer mitochondrial membrane was continuous with the reticular membranes. These cytoplasmic membranes are smooth or studded with ribosomes. These continuities establish an open channel between the endoplasmic reticulum and mitochondria. Similar connections are also found in isolated preparations of mitochondria from the adult guinea pig ovary. The functional significance of these observations are discussed in relation to biochemical studies which demonstrate a transfer of protein from endoplasmic reticulum to mitochondria.

PHALLOIDIN (PHD) is a toxic cyclopeptide in Amanita phalloides1,2. Rats and mice injected with this toxin die in 2–4 h from hepatic necrosis. Dilatation of the endoplasmic reticulum (ER) with the appearance of very large vacuoles are the first ultrastructural changes in hepatocytes3,4. The first biochemical lesion produced by PHD is unknown2. Hepatocytes are the only cells damaged by PHD in mice and rats5,6; Heart fibroblasts7 and bovine kidney cells5 cultured in vitro are not affected by this toxin.

Smooth and rough endoplasmic reticulum and free ribosomes from rat liver each show cyclic-AMP-stimulated protein kinase activity utilizing exogenous substrates. The protein kinases of smooth and rough endoplasmic reticulum can be divided into two classes, one which is extracted by ionic media, and one more firmly attached enzyme fraction which is solubilized by Triton X-100. The protein kinase of free ribosomes is extracted by ionic media. All the three microsomal fractions support an endogenous phosphorylation of proteins which is only slightly stimulated by cyclic AMP. The endogenous phosphorylation shows two pH optima at pH 6.5 and 8.5. The phosphate is incorporated into seryl and threonyl residues of several protein species. Two major phosphoproteins are present in both smooth and rough endoplasmic reticulum, while a third major phosphoprotein is present only in the smooth fraction. There are also several minor phosphoproteins in the two fractions. The endogenous phosphorylation is initially rapid, especially in smooth and rough endoplasmic reticulum where it reaches a maximum after 10–15 min incubation. The endogenously phosphorylated microsomal fractions also support an endogenous dephosphorylation, which is rapid initially, but which leaves approximately 60% of the phosphoryl groups unhydrolyzed. Like the protein kinase the proteins of smooth and rough endoplasmic reticulum which can undergo endogenous phosphorylation can be divided into two classes, one which is extracted in ionic media and one more tightly bound which is solubilized by Triton X-100. Protein kinase, cyclic-AMP-binding material, and protein substrates extracted from smooth and ruogh endoplasmic reticulum by salt or detergent all show recoveries substantially exceeding 100%, suggesting that these activities are partly masked while associated with membrane material.

In spite of the increase in volume of literature on the fine structure of events during division in cells of higher plants, the presence of any kind of a centriole or centriole-like structure has not been demonstrated. However, recent studies have contained data which point to a relationship between membranous elements (usually smooth endoplasmic reticulum) and spindle microtubules in polar regions of dividing cells. These previously reported data, together with data in this present work, are combined into the formulation of an hypothesis relative to the possible role of membranes, particularly smooth endoplasmic reticulum, as cell centers in the assembly and disposition of spindle microtubules in dividing cells of higher plants.

SummaryA characteristic complex of mitochondria and endoplasmic reticulum occurs in the yolk sac epithelium of carnivores and guinea pigs, and in the cytotrophoblastic cells of Langhans in the human placenta. In this complex, ribosomes are located predominantly on the cisternal membrane facing the mitochondrion. The significance of this complex is discussed briefly with respect both to protein synthesis and to fetal physiology.

In cell-free protein synthesis by the murine plasmacytoma X5563, which had become a nonproducing mutant, mixed systems with free polyribosomes and mirosomes incorporated 14 C-amino-acid into protein 3–8 times greater than the sum of the incorporations in the individual system irrespective of S-100 concentrations. This enhancement was inhibited by lecithinase A and was markedly reduced at high KCl concentrations. Smooth endoplasmic membranes had more stimulatory activity than rough endoplasmic membranes. The results indicate that the membrane of the endoplasmic reticulum and free polyribosomes interact in the cell-free protein-synthesizing system, resulting in the enhancement of protein synthesis.

Smooth-surfaced elements of endoplasmic reticulum contact and are attached to the outer membranes of mitochondria in rat liver and onion stem. Some connections appear as short, 150–300 A diameter tubules that bridge the space between the conjoining elements. In liver, the smooth-surfaced endoplasmic reticulum cisternae connected to the outer mitochondrial membrane are shown to be continuous with rough-surfaced endoplasmic reticulum. Here, the smooth-surfaced endoplasmic reticulum is identified in negatively stained preparations of isolated cell fractions and in thin sections of tissues by the presence of lipoprotein particles characteristic of this cell component. In onion, the identification of endoplasmic reticulum is based on continuity with rough-surfaced endoplasmic reticulum.

The cellular changes which lead to the increase of drug-metabolizing enzymes following drug administration are explained by correlating new biochemical data with previously reported electron microscopic and pharmacologic observations. Repeated administration of phenobarbital and several other drugs results in a quantitative increase of the smooth endoplasmic reticulum of the liver cell. The marked increase in drug-metabolizing enzymes is found to occur in this enlarged smooth membrane fraction of the endoplasmic reticulum.

There is a higher activity of ethyl morphine N-demethylase (EM-ase) and cytochrome P-450 (P-450) reductase as well as higher P-450 content in the smooth endoplasmic reticulum (SER) than in the rough endoplasmic reticulum (RER). The extent of the irreversible binding of the 14 C from 14 CCl 4 to lipids and proteins, as well as the CCl 4 -induced destruction of P-450 is more intense in SER than in RER while the opposite was found for glucose 6-phosphatase (G6P-ase) destruction. CCl 4 -induced lipid peroxidation is as intense in SER as is in RER. 14 C from 14 CCl 4 gets irreversibly bound to ribosomal proteins.

The study of gene expression in higher organisms is rendered difficult by the complexity of their cells and tissues. In contrast to prokaryotes, where the translation and transcription of genetic information take place in one compartment, in eukaryotes the two processes are separated topologically by the nuclear membrane. In addition, genetic information in these cells is also present, and is utilized, within certain cytoplasmic organelles, notably chloroplasts and mitochondria, and the operation of the nuclear and extranuclear genomes is coordinated by some yet unknown mechanisms; this topic was the subject of an excellent symposium held here in Australia in late 1969 (cf. Boardman et al., 1971). The association of many enzyme functions with various intracellular membranes also calls for a coordination of protein synthesis within the synthesis of other membrane constituents, primarily phospholipids (Ernster and Orrenius, 1965; Siekevitz et al., 1967; Siekevitz, 1972). A further complexity in multicellular organisms exists in the form of a regulation of gene expression between different cells and tissues by means of environmental, neural and hormonal stimuli (Knox et al., 1956; Tata et al., 1963; Tata, 1967a,b; Greengard, 1970; Knox, 1972).

During electrophoresis on acrylamide gel 30 to 45 percent of the protein of hepatic microsomal membranes migrates as a single band corresponding to a molecular weight of 52,000. Rough and smooth microsomal membranes exhibit essentially identical electrophoretic patterns. Different findings by previous workers may be the result of contamination of the membranes by adsorbed and entrapped nonmembrane protein.

The intracellular site where lysosomal enzymes are revealed by electron histochemical methods appears to be related to the prevailing type of endoplasmic reticulum, i.e., rough- or smooth-surfaced. In rat prostatic and seminal vesicle epithelial cells the rough-surfaced endoplasmic reticulum predominates. Formation of lysosomes and synthesis of at least some of their enzymes appear to be connected with the ribosome-coated membranes of the endoplasmic reticulum. In sebaceous secretory cells and Euglena cells the endoplasmic reticulum is almost exclusively of the smooth-surface variety. In these cells, formation of lysosomes and synthesis of their enzymes appears to be mediated chiefly through the Golgi apparatus. Lysosomes such as described in the various cell types in this work seem to represent “pure lysosomes,” in the sense that they do not appear to be actively engaged in intracellular lytic processes. That they may do so under various physiological and pathological conditions has been indicated in previous reports (2, 3).

Oocytes of the hamster, rat, Mongolian gerbil and squirrel monkey demonstrate an unusual “twin” or multilaminated arrangement of the rough endoplasmic reticulum. Two or more cisternae appose laterally apparently after the ribosomes vanish from the adjacent surfaces. A central electron dense leaflet, composed of filamentous components can be discerned between the apposed surfaces. Between two cisternae of the membrane complex found in hamster oocytes, narrow connecting pillars or walls also develop. The nuclear envelope frequently represents one cisternal element in the membrane complex, at which place nuclear pores are lacking. In rat oocytes the endoplasmic reticulum cisternae take on either a concentric form, apparently by end-to-end fusion of “twin” cisternal membranes, or form a spiral by several turns of the same cisternum. No functional role could be suggested for the observed specilization of the endoplasmic reticulum.

For many years, the cytoplasm of the living cell was conceived of as a rather homogeneous gel in which particles such as the plastids and the mitochondria were suspended. The electron microscope revealed the existence of additional structures in the cytoplasm and clearly established the complex nature of the cytoplasm. Among the first structures observed with the electron microscope were the endoplasmic reticulum, or ER, and the ribosomes.

1 The different biochemical components of endoplasmic reticulum membranes (phospholipids, phosphoproteins and RNA) were investigated as to their ability to interact with the ribosomal protein and RNA. 2 Light-scattering determinations, viscosity measurements and gel-filtration techniques were employed. 3 A complex between a fraction of ribosomal protein (dialyzed against urea) and a phosphoprotein fraction from endoplasmic reticulum membranes, and a complex between a fraction of ribosomal protein (dialyzed against water) and two different phosphoprotein fractions from endoplasmic reticulum membranes, were obtained. No complexes could be detected between the other components under investigation. Likewise no complexes could be detected in the control experiments with lecithin, pepsin and cytochrome c, tested with ribosomal protein fractions and endoplasmic reticulum membranes phosphoproteins. 4 These results suggest an additional new mechanism concerning the interaction of ribosomes with the endoplasmic reticulum membranes.

Continuity of outer mitochondrial membrane with tubular profiles of endoplasmic reticulum (ER) character is found in rat liver hepatocytes and in the ciliateTetrahymena pyriformis. Such membrane continuity is evident from thin sections through intact cells and isolated fractions as well as with negatively stained isolated mitochondria. The ER-profiles continuous with the outer mitochondrial membrane appear predominantly “smooth”, whereas in some cases they showed a few ribosomes associated with them. The observations are discussed as another indication of the close structural and chemical relationship between the outer mitochondrial membrane and the ER. In addition, it is hypothesized that this cisternal continuity provides a route for transfer of special proteins (and possibly lipid components, too) from the rough ER into the mitochondria.

Morphological and biochemical studies indicate that plasma membranes (PM) are derived from nuclear membranes (NM) or endoplasmic reticulum (ER) via the Golgi apparatus (GA) by a process of membrane flow and differentiation (14,16). According to this concept, membranes are transferred and transformed along a chain of cell components in a subcellular developmental pathway.