Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in the nuclear envelope (NE), through which exchange of molecules between the nucleus and cytosol occurs. Biogenesis of NPCs is complex and poorly understood. In particular, almost nothing is known about how NPCs are anchored in the NE. Here, we characterize vertebrate NDC1--a transmembrane nucleoporin conserved between yeast and metazoans. We show by RNA interference (RNAi) and biochemical depletion that NDC1 plays an important role in NPC and NE assembly in vivo and in vitro. RNAi experiments suggest a functional link between NDC1 and the soluble nucleoporins Nup93, Nup53, and Nup205. Importantly, NDC1 interacts with Nup53 in vitro. This suggests that NDC1 function involves forming a link between the NE membrane and soluble nucleoporins, thereby anchoring the NPC in the membrane.

The nuclear pore complex (NPC) is an evolutionarily conserved structure that mediates exchange of macromolecules across the nuclear envelope (NE). It is comprised of ϳ30 proteins termed nucleoporins that are each present in multiple copies. We have investigated the function of the human nucleoporin Nup53, the ortholog of Saccharomyces cerevisiae Nup53p. Both cell fractionation and in vitro binding data suggest that Nup53 is tightly associated with the NE membrane and the lamina where it interacts with lamin B. We have also shown that Nup53 is capable of physically interacting with a group of nucleoporins including Nup93, Nup155, and Nup205. Consistent with this observation, depletion of Nup53 using small interfering RNAs causes a decrease in the cellular levels of these nucleoporins as well as the spindle checkpoint protein Mad1, likely due to destabilization of Nup53-containing complexes. The cellular depletion of this group of nucleoporins, induced by depleting either Nup53 or Nup93, severely alters nuclear morphology producing phenotypes similar to that previously observed in cells depleted of lamin A and Mad1. On basis of these data, we propose a model in which Nup53 is positioned near the pore membrane and the lamina where it anchors an NPC subcomplex containing Nup93, Nup155, and Nup205. INTRODUCTIONThe nuclear envelope (NE) is a specialized membrane system that functions to separate the eukaryotic genome from the cytoplasm. The NE consists of an inner and an outer nuclear membrane. The former contains a distinct set of associated proteins, whereas the latter is structurally equivalent to the endoplasmic reticulum (reviewed in Mattaj, 2004). The nucleoplasmic face of the metazoan inner nuclear membrane is connected to a fibrous protein meshwork termed the nuclear lamina that forms a shell around the underlying chromatin mass. The lamina is composed of Aand B-type lamins, which are closely related to one another and to the intermediate filament-like family of proteins (reviewed in Burke, 2001). The nuclear lamina has been suggested to be involved in maintaining the structural integrity of the NE and influencing chromatin structure and function. The association of the lamina with transcriptionally inactive heterochromatin has led to the suggestion that it may play a role in regulating gene expression and some recent data support this idea (reviewed in Mattout-Drubezki and Gruenbaum, 2003).At numerous points along the NE the inner and outer nuclear membranes fuse to form pores ϳ100 nm in diameter. These pores are occupied by complex macromolecular structures termed nuclear pore complexes (NPCs). The NPCs are associated with euchromatin channels that extend into the interior of the nucleus, interrupting the continuity of the lamina and the heterochromatin. These complex structures have an estimated mass of ϳ60 million Daltons, but are composed of a relatively small number of proteins (ϳ30; Rout et al., 2000;Cronshaw et al., 2002) termed nucleoporins or nups. This is explained by the fact that all of these prot...

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly. INTRODUCTIONThe nuclear envelope (NE) provides a physical barrier between the nucleus and cytoplasm and their unique metabolic tasks. The NE is defined by three morphologically distinct regions. The outer nuclear membrane (ONM) is continuous with the rough endoplasmic reticulum (ER) and contains a similar set of proteins (reviewed in Mattaj, 2004). The inner nuclear membrane (INM) lies adjacent to the nucleoplasm and contains a unique repertoire of proteins that, in part, mediate its interactions with the nuclear lamina and chromatin. Finally, at numerous locations along the NE, the ONM and INM are interrupted by pores where the INM and the ONM are bridged by a connecting membrane termed the pore membrane (POM) domain. Within these pores reside the nuclear pore complexes (NPCs), aqueous channels that provide portals for both passive and active transport of macromolecules.An NPC contains ϳ30 nucleoporins (Nups), many of which are evolutionarily conserved in structure and function (Tran and Wente, 2006). In many cases these Nups are organized into subcomplexes that are present in multiple copies, and they are distributed around the central axis of the NPC, contributing to its characteristic eightfold symmetry. Different groups of Nups and their respective subcomplexes contribute to distinct structural components of the NPC. For example, Nup214/Nup84 are components of the cytoplasmic fibrils (Kraemer et al., 1994;Bastos et al., 1997), the Nup53/Nup155 complex is part of the central core (Marelli et al., 1998), and Nup153 and Tpr contribute to the nucleoplasmic ring and fibrils (Krull et al., 2004).When metazoan cells enter mitosis, their NE is disassembled allowing for the mitotic spindle to access the condensed chromatin. Several models based on previous findings have been proposed to explain the mechanism by which this occurs (reviewed in Hetzer et al., 2005). NE disassembly is accompanied by the phosphorylation of multiple NE proteins including the lamins and several Nups (Burke and Ellenberg, 2002, and refer...