Carl M. Feldherr

Nucleocytoplasmic exchange of macromolecules

Abstract Quantitative measurements of the cytoplasm-to-nucleus exchange of specific protein tracers were correlated with known physical properties (size and electrical charge) of the proteins. Tracers differing in their molecular parameters were produced by fluorescence labelling of wellcharacterized proteins (bovine serum albumin, mol. wt 67 500; ovalbumin, mol. wt 45 000; myoglobin, mol. wt 17 500; lysozyme, mol. wt 14 500; and cytochrome c , mol. wt 13 000) with fluorescein isothiocyanate. The labelled proteins were microinjected into the cytoplasm of living cells, and their uptake into the nucleus was followed by quantitative fluorescence microscopy. In addition, the distribution of cytoplasmically injected ferritin (mol. wt 465 000) was observed with the electron microscope. Five hours after injection, ferritin was localized almost entirely in the cytoplasm and serum albumin remained heavily concentrated in the cytoplasm. Ovalbumin showed significant nuclear uptake within 15 min, but remained more concentrated in the cytoplasm for up to 5 h. Cytochrome c was approximately evenly distributed between cytoplasm and nucleus within 5 min and remained so up to 5 h. Myoglobin and lysozyme each showed significant nuclear concentration above the cytoplasmic level within 3 min. Thus, the cytoplasm-to-nucleus transfer of proteins was limited by their molecular size. Small proteins, neutrally and positively charged, showed rapid nuclear incorporation. Nuclear concentration occurred with some, but not all, small proteins, and was not based simply on the size or charge of the penetrant.

The uptake of endogenous proteins by oocyte nuclei.

Abstract Late stage 5, and stage 6 Xenopus oocytes were micro-injected with tritiated leucine or proline. Incorporation of the isotope into TCA-precipitable material was complete in approx. 1 2 h . At intervals of 1 2 , 1, 3, and 6 h after injection, nuclei were isolated by hand and fixed in ethanol. The nuclear envelopes were removed, and the size distribution of labelled polypeptides within the remaining nucleoplasm was determined on SDS-polyacrylamide gels. Labelled polypeptides ranging in molecular weight from about 11000 to 150000 were present in the nucleus 1 2 h after injection. With increasing time, the number of counts per nucleus increased, as did the proportion of larger polypeptides. Using puromycin, it could be demonstrated that the incorporated macromolecules were taken up from the cytoplasm, and not synthesized in the nucleus. After 3 h the concentration of labelled polypeptides having molecular weights of 94 000 and above was about four times greater in the nucleus than in the cytoplasm. These rates of nuclear accumulation of macromolecules are greater than would have been predicted from previous studies of nuclear permeability utilizing exogenous tracers. Possible explanations for these differences are considered.

Comparison of diverse transport signals in synthetic peptide-induced nuclear transport

Several investigations have demonstrated the ability of synthetic peptides homologous to the nuclear transport signal of simian virus 40 large T antigen to induce the nuclear transport of nonnuclear carrier proteins. To determine the generality of peptide-induced transport, six peptides with sequences derived from four previously identified nuclear transport signals were synthesized and examined for their ability to induce the transport of mouse immunoglobulin G following microinjection into the cytoplasm of mammalian cells. Peptides containing transport signals from simian virus 40 T antigen, Xenopus nucleoplasmin, and adenovirus E1A proteins were highly efficient at peptide-induced transport, while a peptide homologous to yeast MAT alpha 2 protein was incapable of inducing transport. A short nucleoplasmin peptide that contained only the basic amino acid domain was capable of inducing transport but yielded a much slower rate of transport than a long nucleoplasmin peptide encompassing the previously identified minimal transport signal. The short nucleoplasmin signal exhibited a greater capacity for transport than a peptide homologous to the cytoplasmic mutant T antigen signal when conjugates with a low number of signals coupled per carrier protein were examined. However, the short nucleoplasmin peptide was only marginally more effective than the T antigen mutant peptide when conjugates with a high number of signals coupled per carrier protein were examined.

Role of nuclear trafficking in regulating cellular activity.

Publisher Summary This chapter discusses the role of nuclear trafficking in regulating cellular activity. The focus is on the specific and nonspecific mechanisms used by cells to regulate the nucleocytoplasmic distribution of proteins and RNA. Regulation at this level could be achieved either by variations in the transport machinery or the properties of the permeant molecules. Nuclear envelope separates the nucleoplasm from the remainder of the cell. The envelope is a double membrane structure that contains the nuclear pores, circular spaces that are 70-80 nm in diameter, which are formed by the fusion of the inner and outer membranes. The pores are not simply the openings that permit free communication between the nucleoplasm and cytoplasm, but contain highly organized supramolecular protein structures that regulate the movement of macromolecules. The pores, along with their structural elements, are referred to as “pore complexes.” Macromolecular exchanges through the pores can occur either by passive diffusion or signal-mediated transport. The characteristics of the permeant molecules that are most likely to have a major influence on nucleocytoplasmic exchanges relate to signal composition encoded in the primary structure of the protein; the accessibility of the signal to the receptors, which could be affected by either binding reactions that anchor the permeant molecule to cytoplasmic elements or masking of the signal; and post-translational changes, such as phosphorylation, which interfere with signal activity.

Nucleocytoplasmic RNA transport

SummaryA number of closely related post-transcriptional facets of RNA metabolism show nuclear compartmentation, including capping, methylation, splicing reactions, and packaging in ribonucleoprotein particles (RNP). These nuclear ‘processing’ events are followed by the translocation of the finished product across the nuclear envelope. Due to the inherent complexity of these interrelated events,in vitro systems have been designed to examine the processes separately, particularly so with regard to translocation.A few studies have utilized nuclear transplantation/ microinjection techniques and specialized systems to show that RNA transport occurs as a regulated phenomenon. While isolated nuclei swell in aqueous media and dramatic loss of nuclear protein is associated with this swelling, loss of RNA is not substantial, and most studies on RNA translocation have employed isolated nuclei. The quantity of RNA transported from isolated nuclei is related to hydrolysis of high-energy phosphate bonds in nucleotide additives. The RNA is released predominantly in RNP: messenger-like RNA is released in RNP which have buoyant density and polypeptide composition similar to cytoplasmic messenger RNP, but which have distinctly different composition from those in heterogeneous nuclear RNP. Mature 18 and 28S ribosomal RNA is released in 40 and 60S RNP which represent mature ribosomal subunits. RNA transport proceeds with characteristics of an energy-requiring process, and proceeds independently of the presence or state of fluidity of nuclear membranes. The energy for transport appears to be utilized by a nucleoside triphosphatase (NTPase) which is distributed mainly within heterochromatin at the peripheral lamina. Photoaffinity labeling has identified the pertinent NTPase as a 46 kD polypeptide which is associated with nuclear envelope and matrix preparations. The NTPase does not appear to be modulated via direct phosphorylation or to reflect kinase-phosphatase activities. A large number of additives (including RNA and insulin) produce parallel effects upon RNA transport and nuclear envelope NTPase, strengthening the correlative relationship between these activities. of particular interest has been the finding that carcinogens induce specific, long-lasting increases in nuclear envelope (and matrix) NTPase; this derangement may underlie the alterations in RNA transport associated with cancer and carcinogenesis.Two considerations for RNA transport studies are discussed: 1) RNA transportin vitro relates to nuclear swelling, and colloidally-active agents (such as polyvinylpyrrolidone) should be employed to prevent nuclear swelling; and 2) contamination of nuclear preparations by nucleus-absorbed cytoplasmic RNA is substantial, and a detergent-rinse procedure is required to remove this contamination.In considering trafficking of RNP through nuclear pores, conceptual difficulties are obvious. Flux studies with exogenous tracers have indicated patent pore diameters of 90–120 Å. However, the RNP which cross the nuclear envelope (presumably through pores) are all at least 200 Å in diameter. Further, evidence is presented showing that nucleoplasmin-coated gold particles (of diameter 200 Å) pass through central channels in nuclear pores. Whereas previous models of RNP transport have enjoyed the luxury of ascribing deformability (local unfolding) to these RNP, this clearly is not possible for the coated gold particles. These considerations suggest that facilitated translocation of RNP occurs in some manner, and that the mechanisms governing this translocation are not subject to the constraints suffered by exogenous tracers in a ‘rigid-channel’ model of the nuclear pore.

Ribosomal RNA synthesis and transport following disruption of the nuclear envelope

SummaryExperiments were designed to determine if the synthesis, processing and transport of rRNA are affected by changes in nuclear permeability. RNA was labeled by microinjecting [3H]GTP into the cytoplasm of defolliculated oocytes. After injection, the nuclear envelopes were disrupted by puncturing the cells with glass needles. It has been shown that this procedure significantly alters the physical properties of the nuclear envelope. At appropriate intervals after puncturing, the oocytes were manually enucleated and RNA was extracted from both nuclear and cytoplasmic fractions. The extracts were analyzed on 2.5% polyacrylamide gels. The results indicate that over a period of 41/2 h neither the production nor the nucleocytoplasmic distribution of rRNA are affected by altering the permeability characteristics of the nuclear envelope.

The binding characteristics of the nuclear annuli

Abstract The binding of negatively charged colloidal gold particles, coated with bovine serum albumin, to the nuclear pore complex of frog oocytes was investigated under conditions which would alter the electrical charge of the annular material. Extensive binding was observed at pH 7.2, and could be reduced by (1) increasing the pH to 9.1, (2) pretreating the nuclei with 1-fluoro-2,4-dinitrobenzene, or (3) prior digestion of the nuclei with trypsin. It was concluded that the binding of negative particles to the pore complex at physiological pH is due to fixed positive charges associated with protein components of the annuli.

Evidence for changes in nuclear permeability during different physiological states

It was found that the nuclei of well-fed amoebae accumulate colloidal gold from the cytoplasm at a significantly greater rate than the nuclei of cells starved for five or nine days. The results are most likely due to a decrease in the permeability of the nuclear envelope during starvation. Evidence was obtained indicating that the permeability decrease is caused by a change in the functional properties of the pores, rather than a change in either pore size or number. These findings are consistent with the view that the nuclear pores are involved in regulating cellular activity.

The effect of carboxyl-terminal deletions on the nuclear transport rate of rat hsc70

Rat brain hsc70 is a constitutively expressed member of the 70-kDa family of heat shock proteins that is capable of bidirectional transport across the nuclear envelope when microinjected into Xenopus oocytes [1]. The objective of this study was to identify domains involved in its bidirectional transport. Limited proteolytic digestion with chymotrypsin generated three major truncated proteins of approximately 67.5, 59.5, and 56.5 kDa. Reactivity with NH2-terminal-specific antibodies showed that carboxyl-terminal fragments were removed. Nuclear uptake studies were performed by microinjecting 125I-labeled proteins into the cytoplasm and determining their subsequent nucleocytoplasmic distribution. The accumulation rates, while faster than bovine serum albumin controls, were inversely related to the size of the truncated proteins and greatly reduced compared to undigested hsc70. Nuclear efflux was assayed by microinjecting labeled proteins directly into oocyte nuclei. The relative efflux rates of the truncated polypeptides were less than the undigested protein, and, as observed for uptake, were inversely related to size. These results indicate that the carboxyl-terminal domain of hsc70 is involved in its bidirectional exchange.

The effect of temperature on nuclear permeability

Die relative Transportgeschwindigkeit von kolloidalem Gold durch die Membran des Zellkerns verringert sich, wenn die Temperatur von 34° auf 10°C herabgesetzt wird. Ein ähnlicher Temperatureffekt darf für den Transport von Ribonukleoproteinen angenommen werden, da diese Substanzen denselben Weg benützen wie kolloidale Teilchen. Werden die Zellen bei 2°C inkubiert, so machen sich Anzeichen einer Zerstörung der Kernmembrane bemerkbar. Die beobachteten Konzentrationsunterschiede von kolloidalem Gold innerhalb des Kernplasmas konnten nicht durch Grössenänderungen der Zentralkanäle in den Kernporen erklärt werden.