טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMruwat Noor
SubjectMolecular Dissection of Nuclear Pore Complex Assembly
in a Cell-Free Reconstitution System
DepartmentDepartment of Biology
Supervisor Professor Amnon Harel
Full Thesis textFull thesis text - English Version


Abstract

The nuclear pore complex (NPC) is a huge proteinaceous assembly that penetrates the nuclear envelope (NE) and mediates selective bidirectional traffic between the cytoplasm and the nucleus. The NPC is composed of ~30 different subunits called nucleoporins (Nups). The NE of higher eukaryotic cells reforms at the exit from mitosis, in concert with the assembly of NPCs. The earliest step in NPC biogenesis involves the “seeding” of chromatin with ELYS and the Nup107-160 complex. Very little is known about any of the subsequent steps in the assembly pathway.

We study NPC assembly in a cell-free nuclear reconstitution system based on fractionated Xenopus laevis egg extracts. In this work we identify a novel assembly intermediate using egg cytosol manipulated by a truncated form of the nuclear transport receptor Importinβ (Impβ45-462).

Pre-incubation of cytosol with immobilized Impβ45-462 resulted in the reproducible and quantitative removal of the whole set of FG Nups (Nup153, Nup214/CAN, Nup98, Nup62 and Nup358), which are considered to be peripheral components of the mature NPC structure. Nuclei assembled from this Impβ45-462-treated cytosol were not functional for classical NLS-mediated import. Nevertheless, these aborted assembly intermediates were surrounded by intact membranes, like normal nuclei. Rim staining, produced by antibodies directed against FG Nups and which is typical of mature NPCs, was absent in nuclei reconstituted from Impβ45-462-treated cytosol. Nup107 and Nup85, both components of the Nup107-160 complex, are stained more strongly in Impβ45-462 nuclei relative to control nuclei. Anti Nup93 and anti gp210 give similar signal intensities in both types of nuclei, while the transmembrane protein POM121 produces a weak diffuse staining pattern in Impβ45-462 nuclei.

Furthermore, these aborted assembly intermediates were permeated by 9 KDa and 20 KDa dextrans, whereas 155 KDa dextran and anti-DNA antibody molecules were excluded. These selective permeability properties, together with the absence of FG Nups and functional import, suggest that Impβ45-462 nuclei contain a novel kind of initial pore channel that connects the surrounding cytosol and the nuclear interior and is possibly formed by the subset of Nups that are recruited to the NE under these conditions. Our results also imply that the novel assembly intermediates have advanced past a putative fusion step between the outer and inner nuclear membranes.

We can infer that these intermediates contain an early diffusion channel that is not competent in active import. These findings have direct implications for the current mechanistic view of NPC assembly.