Structural and functional characterization of the p62 complex, a subcomplex of the nuclear pore complex

The nuclear pore complex (NPC) is a highly conserved eukaryotic protein complex, which perforates
the nuclear envelope and regulates nucleocytoplasmic transport of cargos between the cytoplasm and nucleus. The structure and function of NPCs were examined in recent years by different molecular and structural biology techniques, such as immunoprecipitation, RNA interference, or electron- and fluorescence microscopy, allowing deeper insights into the molecular mechanisms underlying nucleocytoplasmic
transport. In this introduction, I will highlight recent developments in understanding the organization of four subcomplexes of the central region of the NPC, namely, the p62 complex, the Nup93 complex, the Nup107-160 complex, and the Nup155 complex as well as their impact on nucleocytoplasmic transport. In addition, I will discuss the role of these subcomplexes in cell cycle regulation and their impact
on human diseases. Furthermore, the molecular interactions between different transport receptors, cargos, and components of the NPC are described.
The nuclear pore complex (NPC) is the only known gateway for exchange of macromolecules between the cytoplasm and nucleus of eukaryotic cells. One key compound of the NPC is the p62 subcomplex,
which consists of the nucleoporins p62, p54, and p58/p45 and is supposed to be involved in nuclear protein import and export. In this study we show the localization of different domains of the p62 complex by immuno-electron microscopy using isolated nuclei from Xenopus oocytes. To determine the exact position of the p62 complex, we examined the localization of the C- and N-terminal domains of p62 by immunolabeling using domain-specific antibodies against p62. In addition, we expressed epitope-
tagged versions of p62, p54, and p58 in Xenopus oocytes and localized the domains with antibodies against the tags. This first systematic analysis of the domain topology of the p62 complex within the NPC revealed that the p62 complex is anchored to the cytoplasmic face of the NPC most likely by the coiled-coil domains of the three nucleoporins. Furthermore, we found the phenylalanine-glycine (FG)-repeat domain of p62, but not of p58 and p54, to be mobile and flexible nature.
Nuclear pore complexes (NPCs) are large protein complexes, which are embedded in the nuclear envelope (NE) and control the traffic of proteins and RNAs between the nucleus and the cytoplasm in a signal-dependent manner. Transport receptors bind cargos and interact particularly with certain nucleoporins.
Phenylalanine-glycine (FG) repeat motifs were found in about a third of the nucleoporins, functioning as a major docking site for soluble transport receptors. The p62 complex, which contains several FG repeat domains, was previously described to be involved in protein import and to interact directly with soluble transport receptors. To examine the localization of this docking site and to find out how antibodies against single components of the p62 complex influence nucleocytoplasmic transport, we performed in vitro transport assays using nucleoplasmin-GFP as cargo. This cargo was used in transport assays with digitonin-permeabilized HeLa cells, which were treated with antibodies against the p62 complex components, or was directly conjugated to colloidal gold in ultrastructural transport assays, using isolated nuclei from Xenopus oocytes. Our data suggest that antibodies against the components of the p62 complex inhibit or reduce transport of cargos through the NPC. The ultratructural transport studies revealed that the second docking site for cargo/receptor complexes is masked when p62 complex antibodies are used in the transport assay.
During the past few years, evidence accumulated that distinct nuclear pore complex proteins do not only function in the nucleocytoplasmic transport, but also in the regulation of other cellular processes, such as mitosis. To study the function of the p62-complex (i.e. p62, p54, and p58) in a cellular context, we depleted its components from HeLa cells by RNA interference, which led to an arrest in cell growth and an increase of apoptotic cells as analyzed by fluorescence activated cell sorting. In vitro transport studies of p62- and p54-depleted HeLa cells further showed that the depletion of these p62 complex components had a significant inhibitory effect on nuclear protein import. Taken together, these results indicate that the p62 complex is not only critical for mediating nuclear protein import, but also for cell growth and cell division.
To determine the location of different domains of the p62 complex within the 3D structure of the NPC, we localized the different FG-repeat and coiled-coil domains of this subcomplex with immuno-EM. Previous immuno-EM showed controversial results concerning the anchoring sites and the localization of the p62 complex within the 3D structure of the NPC. Our new data revealed that the p62 complex is anchored with its coiled-coil domains to the cytoplasmic side of the NPC and that its FG-repeat domains show differences in their flexibility and their distribution within the 3D architecture of the NPC. Thus, the FG-repeat domain of the nucleoporin p62 could be found at the cytoplasmic as well as at the nuclear
side of the NPC, whereas the FG-repeat domains of p54 and p58 were restricted to the cytoplasmic side. This might be due to different anchoring sites of the distint FG-repeat domains, the varied length of the FG-repeat domains of the complex, and differences in the biophysical behavior of the FG-repeat domains.
Recently, a complementary technique to immuno-EM was established to observe FG-repeat domains
in vitro by AFM. Lim et al. examined the biophysical behavior of the FG-repeat domain of Nup153, immobilized to a gold dot, by AFM simulating nuclear import by adding importin β.and RanGTP (Lim, Fahrenkrog et al. 2007). The immobilized FG-repeat domains formed a polymer brush with a certain height, which collapsed after addition of importin β. This collapse could be reversed after addition of RanGTP, demonstrating a new biophysical principle for nucleocytoplasmic transport. The AFM-studies were attended by immuno-EM-studies with isolated nuclei of Xenopus oocytes, which showed that, after addition of importin β, the FG-repeat domains of Nup153 collapse to their anchoring sites at the nuclear basket.
It would be interesting to use the same experimental approach equally for the different FG-repeat domains of the p62 complex in order to examine if the FG-repeat domains can form polymer brushes similar to the ones formed by the FG-repeat domains of Nup153. Beyond that, it would be worth to uncover
the anchoring sites of the FG-repeat domains of the p62 complex by immuno-EM after addition of importin β (Lim, Fahrenkrog et al. 2007). In the near future, progress in EM tomography will facilitate the localization of antibodies against certain nucleoporins without the use of gold particles, which would reveal the exact binding sites of the antibodies to structural elements of the NPC.