| dc.description.abstracteng | The nuclear pore complex (NPC) controls the communication between the nucleus and the cytoplasm in a selective manner via a hydrophobic barrier involving phenylalanine-glycine (FG) repeats. Interactions with components of this barrier initiate the transport across the NPC. Nucleoporins are the building unit of the NPC. The NPC is composed of multiple copies of 30 different nucleoporins, which have a defined localization pattern along the nuclear pore complex. Nup214 and Nup88 are two nucleoporins, which exist as a subcomplex at the cytoplasmic face of the nuclear pore.
CRM1 is the main nuclear export receptor of the importin β superfamily that binds to nuclear export sequences (NESs) and mediates the transport of a large number of proteins across the NPC. The crystal structure of a C-terminal fragment of Nup214 and a trimeric export complex containing CRM1, RanGTP and an export cargo points to a specific role of hydrophobic patches in CRM1 that interact with FG-repeats of the nucleoporin.
In an intact cell, Nup214 and its binding partner Nup88 exist as a complex. While Nup214 is a FG-nucleoporin, Nup88 does not have FG repeats. The two proteins were found to be interdependent and the presence of Nup88 is highly dependent on Nup214 with previous results showing loss of Nup88 upon depletion of Nup214, but not the other way around. Interestingly, a putative NES region in Nup214 was identified, which is in close proximity to the Nup88-binding site. Using fluorescence microscopy, the effect of the NES on the localization of Nup214 and Nup88 was investigated. In this context, coordinated overexpression studies of Nup214 and its fragments/mutants with Nup88 were performed. Additionally, biochemical assays were used to form a CRM1/RanGTP-dependent complex with Nup214 NES and FG-repeat fragments. Furthermore, the affinity of Nup214 NES was tested and compared to previously published NESs. RNAi experiments were performed to investigate the role of Nup358 in incorporation of Nup214 into the NPC.
The results revealed that Nup214 contains an NES which has a high binding affinity for CRM1. Additionally, Nup214 can interact with CRM1 in a RanGTP dependent manner and localizes to the cytoplasm and at the nuclear envelope (NE). The CRM1 inhibitor, LMB, shifted the localization of tagged versions of Nup214 to the nucleus and a mutation in the Nup214 NES sequence led to similar results. Co-immunoprecipitation experiments showed that Nup214 and Nup88 can interact independently of the mutations in the NES region of Nup214. Furthermore, knockdown of Nup214 resulted in loss of endogenous Nup88, but the endogenous Nup88 was rescued by re-expression of Nup214. Finally, Nup358 depletion resulted in loss of overexpressed Nup214 at the NE. This study suggests a model of CRM1- and possibly Nup358- dependent Nup214 incorporation into the NPC. | de |