The COPI Complex Functions in Nuclear Envelope Breakdown and Is Recruited by the Nucleoporin Nup153

AbstractNuclear envelope breakdown is a critical step in the cell cycle of higher eukaryotes. Although integral membrane proteins associated with the nuclear membrane have been observed to disperse into the endoplasmic reticulum at mitosis, the mechanisms involved in this reorganization remain to be fully elucidated. Here, using Xenopus extracts, we report a role for the COPI coatomer complex in nuclear envelope breakdown, implicating vesiculation as an important step. We have found that a nuclear pore protein, Nup153, plays a critical role in directing COPI to the nuclear membrane at mitosis and that this event provides feedback to other aspects of nuclear disassembly. These results provide insight into how key steps in nuclear division are orchestrated

LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells

Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope

Defects in nuclear pore assembly lead to activation of an Aurora B–mediated abscission checkpoint

In the absence of nuclear pore components, Aurora B delays abscission to ensure that daughter cells separate only when pores are fully formed

The RNA binding domain within the nucleoporin Nup153 associates preferentially with single-stranded RNA

The nuclear pore protein Nup153 is important for the transport of protein and RNA between the nucleus and cytoplasm. Recently, a novel RNA binding domain (RBD) was mapped within the N-terminal region of Nup153; however, the determinants of RNA association were not characterized. Here we have tested a range of RNAs with different general features to better understand targets recognized by this domain. We have found that the RBD associates with single-stranded RNA with little sequence preference. These results provide new information about a novel RNA binding domain and suggest new models to consider for the contribution of Nup153 to nucleocytoplasmic transport

Changes in nucleoporin domain topology in response to chemical effectors

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The nucleoporin nup153 plays a critical role in multiple types of nuclear export

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin � to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin �/ � or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos

Distinct functional domains within nucleoporins Nup153 and Nup98 mediate transcription-dependent mobility

Despite the apparent overall structural stability of the nuclear pore complex during interphase, at least two nucleoporins have been shown to move dynamically on and off the pore. It is not yet certain what contribution nucleoporin mobility makes to the process of nuclear transport or how such mobility is regulated. Previously, we showed that Nup98 dynamically interacts with the NPC as well as bodies within the nucleus in a transcription-dependent manner. We have extended our studies of dynamics to include Nup153, another mobile nucleoporin implicated in RNA export. In both cases, we found that although only one domain is essential for NPC localization, other regions of the protein significantly affect the stability of association with the pore. Interestingly, like Nup98, the exchange of Nup153 on and off the pore is inhibited when transcription by Pol I and Pol II is blocked. We have mapped the regions required to link Nup98 and Nup153 mobility to transcription and found that the requirements differ depending on which polymerases are inhibited. Our data support a model whereby transcription of RNA is coupled to nucleoporin mobility, perhaps ultimately linking transport of RNAs to a cycle of remodeling at the nuclear pore basket