טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShaulov Lihi
SubjectA Dominant Negative Form of POM121 Binds to Chromatin and
Disrupts the Two Separate Modes of Nuclear Pore
Assembly
DepartmentDepartment of Biology
Supervisor Professor Amnon Harel
Full Thesis textFull thesis text - English Version


Abstract

Nuclear pore complexes (NPCs) are formed during two separate stages of the metazoan cell cycle. NPCs assemble into the re-forming nuclear envelope (NE) at the exit from mitosis and into an intact, expanding NE during interphase. The NPC is one of the largest known macromolecular structures within cells and thought to be structurally and functionally similar in all eukaryotes. POM121 is one of the 3 proteins that are integral membrane nucleoporins. POM121 has a single membrane-spanning segment close to its N-terminus and the bulk of the protein facing the NPC channel, thus potentially interact with a verity of nucleoporins. POM121 orthologues have only been identified in vertebrates. Here, we show that a soluble internal fragment of the membrane nucleoporin POM121 has a dominant negative effect on both modes of assembly in a cell-free reconstitution system.

In this study we use the in vitro nuclear reconstitution assay, which is based on Xenopus egg extracts. We cloned a fragment of POM121 and added it to the nuclear reconstitution as a soluble, dominant negative form of POM121 to the endogenous protein. We found out that the soluble POM121 fragment, termed POM121DN, binds chromatin and prevents membrane enclosure and NPC formation. POM121DN binding does not prevent the seeding of chromatin by ELYS/Nup107-160 subcomplex. The depletion of ELYS abolished Nup107-160 subcomplex binding to chromatin, yet POM121DN was still bound strongly to the chromatin periphery. Thus, the binding sites of POM121 to chromatin are distinct from ELYS/Nup107-160 “seeding” sites. Interestingly, we saw that Importin β negatively regulates chromatin binding by the POM121 fragment via a conserved NLS motif. The inhibition of POM121DN binding to chromatin by Importin β is reversible by addition of RanGTP. The conserved NLS appears to mediate the Importin β-interactions of POM121DN, but does not account in itself for the dominant negative effect on NPC assembly. Our results suggest that Importin  regulates two independent chromatin binding modules: the membrane-anchored POM121 and the soluble ELYS/Nup107-160 subcomplex.

When an intact NE is present before the addition of the POM121 fragment, NPCs are inserted into the NE but membrane expansion is inhibited. This results in densely packed NPCs with no intervening membrane patches. The dominant negative POM121DN fragment completely blocks off NE expansion in the interphase assembly mode and does so independently from the NLS-mediated interaction with Importin  and . We conclude that POM121 plays a critical role in both modes of assembly and links nuclear membrane formation and expansion to nuclear pore biogenesis.