Nuclear protein migration involves two steps: Rapid binding at the nuclear envelope followed by slower translocation through nuclear pores

doi:10.1016/0092-8674(88)90403-5

Cell

Volume 52, Issue 5, 11 March 1988, Pages 655-664

https://doi.org/10.1016/0092-8674(88)90403-5 Get rights and content

Abstract

When injected into the cytoplasm of Vero cells, nucleoplasmin rapidly concentrates in a narrow layer around the nuclear envelope and then accumulates within the nucleus. Transport into the nucleus can be reversibly arrested at the perinuclear stage by metabolic inhibitors or by chilling. Nucleoplasmin-coated colloidal gold particles concentrate around the nuclear envelope of Vero cells or Xenopus oocytes, and by electron microscopy of oocytes appear to be associated with fibrils attached to nuclear pore complexes. Perinuclear accumulation is not observed for the nonmigrating nucleoplasmin core fragment or nonnuclear proteins. We propose two steps in nuclear migration of proteins: rapid binding around the nuclear envelope, possibly to pore-associated fibrils, followed by slower, energy-dependent translocation through nuclear pores.

References (32)

L.I. Davis et al.
Identification and characterization of a nuclear pore complex protein
Cell
(1986)
S.M. Dilworth et al.
Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts
Cell
(1987)
C. Dingwall et al.
A polypeptide domain that specifies migration of nucleoplasmin into the nucleus
Cell
(1982)
W.C. Earnshaw et al.
Assembly of nucleosomes: the reaction involving X. laevis nucleoplasmin
Cell
(1980)
C.M. Feldherr
The binding characteristics of the nuclear annuli
Exp. Cell Res.
(1974)
W.W. Franke et al.
The ultrastructure of the nuclear envelope of amphibian oocytes: a reinvestigation. 1. The mature oocyte
J. Ultrastruct. Res.
(1970)
A. Graessmann et al.
Microinjection of early SV40 DNA fragments and T antigen
Meth. Enzymol.
(1980)
J.A. Kleinschmidt et al.
Co-existence of two different types of soluble histone complexes in nuclei of Xenopus laevis oocytes
J. Biol. Chem.
(1985)
G. Krohne et al.
A major soluble acidic protein located in nuclei of diverse vertebrate species
Exp. Cell Res.
(1980)
R.A. Laskey et al.
Assembly of SV40 chromatin in a cell-free system from Xenopus eggs
Cell
(1977)

C. Schneider et al.

A one-step purification of membrane proteins using a high efficiency immuno matrix

J. Biol. Chem.

(1982)

S. Wischnitzer

An electron microscopy study of the nuclear envelope of amphibian oocytes

J. Ultrastruct. Res.

(1958)

G. Danscher

Localization of gold in biological tissue

Histochemistry

(1981)

C. Dingwall et al.

Protein import into the cell nucleus

Ann. Rev. Cell Biol.

(1986)

C. Dingwall et al.

Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals

EMBO J.

(1987)

C.M. Feldherr

The nuclear annuli as pathways for nucleocytoplasmic exchanges

J. Cell Biol.

(1962)

Cited by (447)

Macromolecular Communication Between Nucleus and Cytoplasm
2022, Encyclopedia of Cell Biology: Volume 1-6, Second Edition
Bidirectional transport of macromolecules occurs through nuclear pores, which were first detected by electron microscopy. Interest in regulation of gene expression, curiosity to understand one of the few remaining uncharacterized translocons of eukaryotic cells, and the hope of ultimate therapeutic relevance have led to a period of intense investigation of these transport pathways. This article outlines the principal features of nucleocytoplasmic transport, highlights less well-understood related issues, and gathers thoughts pertaining to the evolutionary origin of the equipment that accomplishes transport. During the past half century, this field has added a major dimension to the understanding of what it means to be eukaryotic.
Methods to separate nuclear soluble fractions reflecting localizations in living cells
2021, iScience
To understand various intranuclear functions, it is important to know when, what, and how proteins enter the nucleus. Although many methods and commercial kits for nuclear fractionation have been developed, there are still no methods for obtaining a complete nuclear proteome. Soluble nuclear proteins are often lost during fractionation. We developed remarkably improved methods to obtain nuclear soluble fractions by optimizing the conditions of selective permeabilization of the plasma membrane. As a result, 10 million cells could be separated into the cytoplasmic and nuclear soluble fractions more precisely in a 1.5-mL test tube. Moreover, the addition of an inhibitor to prevent leakage from the nucleus retained small proteins in the nucleus. Because of the simple protocols and easy application for multiple samples, our methods are expected to be applied to various studies on spatiotemporal changes of dynamic nuclear proteins, such as signal transduction.
Intracellular delivery of colloids: Past and future contributions from microinjection
2018, Advanced Drug Delivery Reviews
Citation Excerpt :
Functionalized gold (Au) NP have been used in combination with electron or fluorescence microscopy to investigate subcellular organization [46, 119]. In a previously mentioned study where the two-step mechanism of nuclear entry was described for the first time [46], functionalized Au NP constituted a key tool. The attachment of the chaperone protein nucleoplasmin on the surface of the NP targeted them to the nuclear membrane, allowing observations that led to the above-described mechanism of nuclear entry [46].
The manipulation of single cells and whole tissues has been possible since the early 70’s, when semi-automatic injectors were developed. Since then, microinjection has been used to introduce an ever-expanding range of colloids of up to 1000 nm in size into living cells. Besides injecting nucleic acids to study transfection mechanisms, numerous cellular pathways have been unraveled through the introduction of recombinant proteins and blocking antibodies. The injection of nanoparticles has also become popular in recent years to investigate toxicity mechanisms and intracellular transport, and to conceive semi-synthetic cells containing artificial organelles. This article reviews colloidal systems such as proteins, nucleic acids and nanoparticles that have been injected into cells for different research aims, and discusses the scientific advances achieved through them. The colloids' intracellular processing and ultimate fate are also examined from a drug delivery perspective with an emphasis on the differences observed for endocytosed versus microinjected material.
AFM visualization of sub-50 nm polyplex disposition to the nuclear pore complex without compromising the integrity of the nuclear envelope
2016, Journal of Controlled Release
Citation Excerpt :
The translocation of unpacked DNA from the cytoplasm to the nucleus is also debatable; it is expected to be slow and inefficient, since DNA is rapidly degraded in the cytoplasm with an apparent half-life of 50–90 min [15]. Nuclear entry, however, is a selective process tightly modulated by the nuclear pore complex (NPC) [16,17]. NPCs are supramolecular assemblies of nucleoporins displaying a ≤ 39 nm wide transport channel that mediates passive diffusion of molecules up to 40 kDa in size as well as facilitating selective transport of larger molecules bound to soluble transport receptors that shuttle between the cytoplasm and nucleus in eukaryotic cells [18,19].
It has been questioned as to whether polyplexes in the cytoplasm can reach the nuclear compartment and if so in what form. By applying atomic force microscopy (AFM) to the nuclear envelope and the nuclear pore complexes, we demonstrate that disposition of polyethylenimine (PEI)/DNA polyplexes that were microinjected into the oocytes of Xenopus laevis, as an example of a non-dividing cell, is exclusive to the nuclear pore complex (NPC). AFM images show NPCs clogged only with sub-50 nm polyplexes. This mode of disposition neither altered the morphology/integrity of the nuclear membrane nor the NPC. AFM images further show polyplexes on the nucleoplasmic side of the envelope, presumably indicating species in transit. Transmission electron microscopy studies of ruptured nuclei from transfected human cell lines demonstrate the presence of sub-50 nm particles resembling polyplexes in morphology compared with control preparations.
An insertion in the methyltransferase domain of P. falciparum trimethylguanosine synthase harbors a classical nuclear localization signal
2016, Molecular and Biochemical Parasitology
Many Plasmodium falciparum proteins do not share homology with, and are generally longer than their respective orthologs. This, to some extent, can be attributed to insertions. Here, we studied a P. falciparum RNA hypermethylase, trimethylguanosine synthase (PfTGS1) that harbors a 76 amino acid insertion in its methyltransferase domain. Bioinformatics analysis revealed that this insertion was present in TGS1 orthologs from other Plasmodium species as well. Interestingly, a classical nuclear localization signal (NLS) was predicted in the insertions of primate parasite TGS1 proteins. To check whether these predicted NLS are functional, we developed an in vivo heterologous system using S. cerevisiae. The predicted NLS when fused to dimeric GFP were able to localize the fusion protein to the nucleus in yeast indicating that it is indeed recognized by the yeast nuclear import machinery. We further showed that the PfTGS1 NLS binds to P. falciparum importin-α in vitro, confirming that the NLS is also recognized by the P. falciparum classical nuclear import machinery. Thus, in this study we report a novel function of the insertion in PfTGS1.
Macromolecular Communication between Nucleus and Cytoplasm
2016, Encyclopedia of Cell Biology
Bidirectional transport of macromolecules occurs through nuclear pores, which were first detected by electron microscopy. Interest in regulation of gene expression, curiosity to understand one of the few remaining uncharacterized translocons of eukaryotic cells, and the hope of ultimate therapeutic relevance have led to a period of intense investigation of these transport pathways. This article outlines the principal features of nucleocytoplasmic transport, highlights less well-understood related issues, and gathers thoughts pertaining to the evolutionary origin of the equipment that accomplishes transport. During the past half century, this field has added a major dimension to the understanding of what it means to be eukaryotic.

View all citing articles on Scopus

View full text

Cell

Nuclear protein migration involves two steps: Rapid binding at the nuclear envelope followed by slower translocation through nuclear pores

Abstract

Cell

Cell

Cell

Cell

Exp. Cell Res.

J. Ultrastruct. Res.

Meth. Enzymol.

J. Biol. Chem.

Exp. Cell Res.

Cell

J. Biol. Chem.

J. Ultrastruct. Res.

Localization of gold in biological tissue

Histochemistry

Protein import into the cell nucleus

Ann. Rev. Cell Biol.

Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals

EMBO J.

The nuclear annuli as pathways for nucleocytoplasmic exchanges

J. Cell Biol.