Isolation of Two Distinct Classes of Polysomes From a Nuclear Fraction of Rat Liver

175

Electron microscope examination of isolated rat liver nuclei after treatment with the detergent Triton X-100 revealed the complete removal of both the inner and outer membranes of the nuclear envelope. The envelope-denuded nuclei did not show any change in either shape or internal ultrastructure. Most strikingly, the nuclear pore complexes, which in untreated nuclei appear to be integral components of the nuclear envelope, were retained in their characteristic location at the distal ends of the channels leading through the peripheral heterochromatin.Determination of the chemical composition of detergent-treated nuclei showed that over 95% of the nuclear phospholipid was solubilized, thus corroborating the morphological absence of nuclear membranes. Furthermore, detergent treatment also solubilized approximately 10% of the nuclear protein. Analysis of the solubilized protein by polyacrylamide gel electrophoresis in the presence of SDS indicated that these proteins belong to a few specific classes which presumably represent the major polypeptides of the nuclear membranes.The total absence of the nuclear envelope on both morphological and biochemical grounds supports the idea that the nuclear pore complex does not require the membranes either for attachment to the nucleus or for maintenance of its own structural integrity. INTRODUCTIONIn eukaryotes, the nuclear pore complex is a ubiquitous organelle situated within the characteristic circular discontinuities ("pores") of the nuclear envelope (1-9). The roles which have been postulated for the pore and the complex fall primarily into two mutually compatible classes: (a) involvement in nucleo-cytoplasmic communication (9-12), and (b) organization of the interphase chromatin (13 16). Little direct evidence has been obtained to support either role, and there is only fragmentary information concerning the chemical composition of the complex (17, 18).Before attempting the isolation and chemical characterization of the pore complex, it was necessary to determine the structural relationships between the pore complex and its surrounding nuclear membranes and between the pore complex and the underlying peripheral chromatin.Nuclear pore complexes have been described in nuclear envelope fractions (19)(20)(21)(22)(23)(24)(25)(26). Their presence suggests that the bulk of the peripheral chromatin is not necessary for maintaining the gross structural integrity of the complex. However, significant amounts ofchromatin are routinely recovered 746

Section: Discussionmentioning

confidence: 99%

On the Attachment of the Nuclear Pore Complex

Aaronson

Blobel

1974

175

“…VoLumE 46, 1970 • pages [379][380][381][382][383][384][385][386][387][388][389][390][391][392][393][394][395] clear envelope fragments were isolated from mammalian liver first by the method of Franke (26,27) which uses a combination of hypotonic shock and sonication for the nuclear disruption (15,(26)(27)(28)(29)95) and, recently, by other authors employing similar methods (9, 43,97) . Other attempts for obtaining nuclear membrane material from diverse mammalian cells, such as HeLa cells, calf thymus, and rat liver, involved the use of detergents such as deoxycholate and Triton X-100 (4, 75,92,93) which obviously can cause considerable membrane dissociation and thus progressive loss of membrane material, especially in the outer nuclear membrane (40), and consequently have a very limited range of application .…”

Section: Introductionmentioning

confidence: 99%

Nuclear Membranes From Mammalian Liver

Franke

Deumling

Ermen

et al. 1970

190

Nuclear membranes were isolated from rat and pig liver by sonication of highly purified nuclear fractions and subsequent removal of adhering nucleoproteins in a high salt medium . The fractions were examined in the electron microscope by both negative staining and thin sectioning techniques and were found to consist of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents still could frequently be recognized . The chemical composition of the nuclear membrane fractions was determined and compared with those of microsomal fractions prepared in parallel . For total nuclei as well as for nuclear membranes and microsomes, various enzyme activities were studied . The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum, with respect to their RNA : protein ratio and their content of polar and nonpolar lipids . Both membranous fractions had many proteins in common including some membrane-bound enzymes . Activities in Mg-ATPase and the two examined cytochrome reductases were of the same order of magnitude . The content of cytochrome b 5 as well as of P-450 was markedly lower in the nuclear membranes . The nuclear membranes were found to have a higher buoyant density and to be richer in protein . The glucose-6-phosphatase and Na-K-ATPase activities in the nuclear membrane fraction were very low. In the gel electrophoresis, in addition to many common protein bands, some characteristic ones for either microsomal or nuclear membranous material were detected . Significant small amounts of DNA and RNA were found to remain closely associated with the nuclear envelope fragments . Our findings indicate that nuclear and endoplasmic reticulum membranes which are known to be in morphological continuity have, besides a far-reaching similarity, some characteristic differences .

“…The lumen of the endoplasmic reticulum is contiguous with the perinuclear space; therefore, any protein which, like the secretory proteins, moved directly from the site of synthesis into the lumen of the endoplasmic reticulum might enter the nucleus without passing through the soluble space of the cytoplasm. It should be noted that the outer nuclear membrane is studded with polysomes, and that they differ from other cytoplasmic polysomes with respect to the kinds of protein which they synthesize (23) and with respect to their labeling kinetics following a pulse of a radioactive amino acid (23) or RNA precursor (24,25). Studies by Gorovsky using Tetrahymena indicate that the outer membrane ribosomes may be responsible for synthesis of nuclear proteins (23).…”

Section: Discussionmentioning

confidence: 99%

“…Animals were kept on a schedule of alternating periods of light (14 hr) and dark (10 hr) and were given food and water ad libitum until [24][25][26] hr before injection of radioisotope, at whichtime they were deprived of food. All injections were given at the same time of day.…”

Section: In Vivo Labeling Of Liver Proteinsmentioning

confidence: 99%

Studies on the Site of Synthesis of Several Soluble Enzymes of the Cell Nucleus

Kuehl

Sumsion

1971

A B S T R A C TRats were given radioactive L-leucinc intravcnously. At various timcs after injcction, the livers were removed and separated into nuclear and cytoplasmic fractions by a nonaqueous technique. Glyccraldehydc-3-phosphate dehydrogenase, aldolase, and lactic dehydrogenase were isolated from each cell fraction by antibody precipitation followed by gel clectrophorcsis, and the spccific radioactivities of the isolated enzymes were determined. In all three cases, the onset of labeling and the rate of incorporation were thc same for the nuclear enzyme as for the corresponding enzyme from the cytoplasm. If wc assume that equilibration of the enzymes between the cytoplasmic and nuclear pools occurs slowly rclativc to the labeling times employed, we may conclude that the labeled nuclcar enzymes either were synthesized in the nucleus or moved into the nucleus from a cytoplasmic site of synthesis without first passing into the cytoplasmic pool of enzyme. Treatment with puromycin, an antibiotic which depresses incorporation into cytoplasmic proteins to a greater extent than into nuclear proteins, led to a situation in which the specific activities of the nuclear enzymes were several times as high as those of the corresponding cytoplasmic enzymes following a short pcriod of incorporation. Thcse data substantiate the assumption that equilibration between the cytoplasmic and nuclear enzyme pools occurs slowly and provide further evidence that the labeled nuclear enzymes do not arise from the cytoplasmic enzyme pool.