Peptidyl-puromycin synthesis by free and membrane-bound ribosomes.

The peptidyl transferase reaction, as measured by the formation of peptidyl-puromycin, was compared for free ribosomes and ribosomes bound to two types of membrane, the endoplasmic reticulum and the outer nuclear membrane. In most respects the reaction catalyzed by the three types of ribosome was similar, demonstrating that interaction of the 60 S ribosomal subunit with the membrane has little effect on the functioning of peptidyl transferase, a 60 S protein. However, both the rate and extent of synthesis of peptidyl puromycin were lower for ribosomes bound to the nuclear membrane than for free or microsome-bound ribosomes. This difference appears to be a direct consequence of the ribosome-membrane interaction, since ribosomes stripped from the nuclear membrane could not be distinguished from the other classes of ribosome.     

Protein synthesis by membrane-bound and free ribosomes of the developing rat cerebral cortex

1. Rates of RNA and protein synthesis were measured in rat cerebral-cortex slices, and compared with amino acid incorporation into protein by membrane-bound and free ribosomes from the same tissue, in the first 3 weeks of life. 2. A rapid age-dependent decline in the incorporation of labelled precursors into both RNA and protein was observed, which was more marked for amino acid incorporation into protein. 3. Although membrane-bound ribosomes comprise only a small fraction of total ribosomes, they were more active in incorporating amino acids into protein than were free ribosomes, especially immediately after birth. The decline in activity with age was more marked in the membrane-bound fraction than in free ribosomes. This loss of activity was largely independent of alterations in soluble factors or endogenous mRNA content and appeared to involve some alteration of the function of the ribosome itself, with relatively small alterations in the ratio of membrane-bound to free ribosomes. 4. Thyroidectomy, performed soon after birth, had no effect on the incorporation of radioactive precursors into RNA or protein by either slices or the cell-free preparations during the first 3-4 weeks of life.     

Comparison of synexin isotypes in secretory and non-secretory tissues

Three synexin isotypes were identified in bovine liver or adrenal medullary tissues by immune blotting of one- or two-dimensional SDS gels and by two-dimensional tryptic peptide mapping of gel bands or spots. These isotypes were: alpha-synexin, mass 47 kDa, pI 6.9; beta-synexin, mass 47 kDa, pI 6.5; and mu-synexin, mass 51 kDa, pI 6.1. A non-secretory tissue, bovine skeletal muscle, was found to contain only mu-synexin. The absence of alpha- and beta-synexins in a non-secretory tissue suggests these proteins may perform specific roles in the process of exocytosis.     

Protein synthesis by single ribosomes

The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.     

Distribution of membrane-bound and free ribosomes in growing yeast.

1. The distribution of membrane-bound and free ribosomes was investigated in stationary as well as in growing yeast cells. the relative amount of free ribosomes varies with the growth phase of the cell culture. During the duplication phases of the cell, relative maxima of free ribosomes can be found. However, the absolute amount of free ribosomes is fairly constant during the growth of the cells. 2. Membrane-bound ribosomes show lower polypeptide synthesis activity in a cell-free, poly (U)-dependent system than free ribosomes. 3. There is no difference in the distribution pattern of free and membrane-bound ribosomes in growing yeast cells of different ploidy. 4. A turnover between free and membrane-bound ribosomes is suggested to be in agreement with the hypothesis of Branes and Pogo ((1975) Eur. J. Biochem. 54, 317-328).     

The relation of protein synthesis to the concentrations of free and membrane-bound ribosomes in brain at different ages

Free and membrane-bound ribosomes were prepared from the brains of young (3- and 8-day-old) and adult (30 day) rats by the method of Ramsey and Steele (1977). Though the concentration of RNA in young brain is higher than that in adult brain, the fraction of the RNA which is ribosomal is virtually the same (64%) as is the ratio of free ribosomes total ribosomes (61%) at all ages studied. The rate of protein synthesis measured in vivo, expressed in the usual terms of “% per h”, is much higher in young compared to adult brain, but when expressed as the ribosomal specific activity, i.e. “mg protein synthesized per hour per mg ribosomal RNA”, is the same in the three age groups (0.61, 0.58 and 0.60, respectively). Thus, even during early development, when protein is increasing rapidly, ribosomes are no more active than in adult brain, suggesting that synthesis rates in brain are limited by ribosomal content.     

Changes in free and membrane-bound ribosomes during the development of chick liver

A major difficulty in studying quantitative changes in free and membrane-bound ribosomes in a tissue under different physiological conditions is that membrane-bound ribosomes are not usually recovered quantitatively in a conventional microsomal fraction. This problem was resolved for developing chick liver by determining the conditions for the isolation of a microsomal fraction containing the highest practicable yield of rough vesicles, and then separating it into free-ribosome- and rough-vesicle-containing fractions. With the aid of a marker enzyme for the microsomal membranes and the RNA content of the recovered membrane-bound ribosomes, it was possible to correct for the recovery of rough vesicles and hence to determine the concentration of membrane-bound ribosomes in the homogenate. Despite the fact that morphological studies have suggested that most of the cellular ribosomes are not bound to membrane in chick liver cells at the earliest developmental age studied (6 days of egg incubation), 49% of the total ribosomes were found to be membrane-bound by using the new fractionation technique. This fraction increased (to 66%) during development. The discrepancy between the cell-fractionation and morphological approaches could not be attributed to artifacts of the separation method but rather to difficulties inherent in the morphological approach.     

Distribution of free and membrane-bound ribosomes after corticosterone administration in perfused rat liver

Contrasting results have been reported on the effect of steroid hormones on the interaction between ribosomes and endoplasmic reticulum in rat liver. Exposure to high gravitational forces for a long time was found necessary to obtain a constant ratio of free to membrane-bound ribosomes from the post-nuclear supernatant. Using these isolation conditions, rat liver from fasted controls and from fasted, adrenalectomized rats contain both about 45% membranebound ribosomes. Addition of corticosterone to the livers from adrenalectomized rats did not increase the pool of bound ribosomes more than in the control livers; 55% was found in both. Corticosterone had therefore no effect on the pool sizes of free and membrane-bound ribosomes in perfused rat livers.     

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of NADPH-adrenodoxin reductase and adrenodoxin of bovine adrenal cortex mitochondria

Cytoplasmic free and membrane-bound ribosomes were isolated from bovine adrenal cortex, and characterized. Contributions of free and bound ribosomes to the synthesis of NADPH-adrenodoxin reductase (AdR) and adrenodoxin (Ad) were determined by examining the presence of their nascent peptides on isolated ribosomes. Nascent peptides were released from the ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of AdR and Ad were separately isolated by immunoprecipitation using antibodies. AdR nascent peptides were associated with free and loosely-bound ribosomes, whereas Ad nascent peptides were associated with free, loosely-bound and tightly-bound ribosomes. Smaller nascent peptides of AdR were carried by free ribosomes, whereas larger nascent peptides were preferentially carried by loosely-bound ribosomes. In the case of Ad, smaller nascent peptides were more abundant in free ribosomes than in bound ribosomes. The nascent peptides of Ad were released from bound ribosomes of rough microsomes to the aqueous milieu by puromycin treatment, suggesting the release of completed Ad peptides into the cytoplasm in cells.