Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm

Abstract

Bidirectional transport of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complexes (NPCs) by a signal-mediated mechanism that is directed by targeting signals (NLSs) residing on the transported molecules or "cargoes." Nuclear transport starts after interaction of the targeting signal with soluble cellular receptors. After the formation of the cargo-receptor complex in the cytosol, this complex crosses the NPC. Herein, we use gold particles of various sizes coated with cargo-receptor complexes to determine precisely how large macromolecules crossing the NPC by the signal-mediated transport mechanism could be. We found that cargo-receptor-gold complexes with diameter close to 39 nm could be translocated by the NPC. This implies that macromolecules much larger than the assumed functional NPC diameter of 26 nm can be transported into the karyoplasm. The physiological relevance of this finding was supported by the observation that intact nucleocapsids of human hepatitis B virus with diameters of 32 and 36 nm are able to cross the nuclear pore without disassembly.

Figure 1

Electron microscopy of representative gold particles and their protein coat. Gold particles (A, 22 nm; B, 26 nm; and C, 36 nm) uncoated (first row) and coated with NP or BSA (second row) were visualized in the electron microscope by negative staining. The third and fourth rows are examples of NP- and BSA-coated gold particles that were incubated first with importin α (NP + α and BSA + α) and then with importin β (NP + α + β and BSA + α + β). The last row shows examples of NP- and BSA-coated gold particles that were incubated with importin α and β and then immunogold labeled with an anti-importin β antibody directly conjugated with 8-nm gold particles.

Figure 2

Nuclear import of NP-coated gold particles into Xenopus oocyte nuclei. Gold particles of 22 ± 2 nm (A), 26 ± 3 nm (B), and 36 ± 4 nm (C) in diameter were coated with NP and their nuclear import was followed by electron microscopy 1 h after injection into the cytoplasm of Xenopus oocytes. Shown are views of nuclear envelope cross sections with adjacent cytoplasm (c) and nucleoplasm (n). Arrowheads point to gold particles that reached the nucleus or are associated with the nuclear face of the NPC. Gold particles 28 nm in diameter or smaller (without the protein coat) were imported into the nucleus. Bar, 100 nm.

Figure 3

Histograms on the size distribution of gold particles available for import (total), and found within the cytoplasm and the nucleus. Twenty-two ± 2 nm-gold particles, coated with NP (A), 26 ± 3 nm-gold particles, coated with NP (B), 22 ± 2 nm-gold particles, coated with NP and importin α and β (C), and 26 ± 3 nm-gold particles, coated with NP and importin α and β (D). For comparison, only 100 particles in the nucleus and 100 particles in the cytoplasm were measured. The data were derived from microinjections in four different oocytes.

Figure 4

Nuclear import of NP-importin α- and β-coated gold particles into Xenopus oocyte nuclei. NP-coated gold particles with diameter of 26 ± 3 nm were incubated in vitro first with importin α and then with importin β. The resulting complexes were injected into the cytoplasm of Xenopus oocytes and their nuclear import was followed by electron microscopy. Shown are low- (A) and high (B)-magnification views of nuclear envelope cross sections with adjacent cytoplasm (c) and nucleoplasm (n). Arrowheads point to gold particles that reached the nucleus or are associated with the nuclear face of the NPC. Gold particles as large as 26 nm in diameter (without the protein coat) were able to cross the nuclear pores and enter the nucleus of Xenopus oocytes. Shown is also a gallery of gold particles that were found in the nucleus of Xenopus oocytes. Bars, 200 nm (A) and 50 nm (B).

Figure 5

Intact hepatitis B virus cores (32 or 36 nm in diameter) are able to cross the nuclear pores. (A) Views of a nuclear envelope cross section with adjacent cytoplasm (c) and nucleoplasm (n) from a Xenopus oocyte that have had phosphorylated recombinant HBV cores microinjected into their cytoplasm. Arrowheads point to core particles associated with the nuclear face of the NPC. (B) Nuclear envelope cross section from a control (noninjected) Xenopus oocyte. (C) Examples of cross-sectioned NPCs with associated HBV cores. (D) Same micrographs as in C but with the nuclear membrane boundaries and the HVB cores outlined. (E) Quantitative analysis of the size of core particles associated with the cytoplasmic face of the nuclear pore (cNPC) or the nuclear face of the nuclear pore (nNPC) from oocytes injected with recombinant HBV cores. 60 HBV cores were measured for each histogram. Bars, 100 nm.