bims-nucpor 2022-09-04 papers

mBio. 2022 Aug 30. e0181522

TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei.

Sushma V Ambekar, Josh R Beck, Gunnar R Mair.

Twenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium nuclear pore complex (NPC) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here, we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the P. berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry, we identified 4 known nucleoporins (Nups) (138, 205, 221, and the bait 313), and verify interaction with the putative phenylalanine-glycine (FG) Nup637; we assigned 5 proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by green fluorescent protein (GFP) tagging. Based on gene deletion attempts, all new Nups - Nup176, 269, 335, 390, and 434 - are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit 2 incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC (Structural Maintenance Of Chromosomes 4). The protocols developed here showcase the power of proximity labeling for elucidating protein complex composition and annotation of taxonomically restricted genes in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position. IMPORTANCE The nuclear pore complex (NPC) is a platform for constant evolution and has been used to study the evolutionary patterns of early-branching eukaryotes. The Plasmodium NPC is poorly defined due to its evolutionary divergent nature making it impossible to characterize it via homology searches. Although 2 decades have passed since the publication of the Plasmodium genome, 30% of the genes still lack functional annotation. Our study demonstrates the ability of proximity labeling using TurboID to assign function to orphan proteins in the malaria parasite. We have identified a total of 10 Nups that will allow further study of NPC dynamics, structural elements, involvement in nucleocytoplasmic transport, and unique non-transport functions of nucleoporins that provide adaptability to this malaria parasite.

Keywords: BioID; Nup; Plasmodium; TurboID; malaria; nuclear pore complex; nucleoporins; proximity labeling

DOI: https://doi.org/10.1128/mbio.01815-22

Nat Commun. 2022 Sep 01. 13(1): 5138

Percolation transition prescribes protein size-specific barrier to passive transport through the nuclear pore complex.

David Winogradoff, Han-Yi Chou, Christopher Maffeo, Aleksei Aksimentiev.

Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex's pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of experimentally derived NPC structures with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein transport reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistics of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

DOI: https://doi.org/10.1038/s41467-022-32857-1

Front Microbiol. 2022 ;13 895779

Nucleoporin 85 interacts with influenza A virus PB1 and PB2 to promote its replication by facilitating nuclear import of ribonucleoprotein.

Yue-Huan Ling, Hao Wang, Mei-Qing Han, Di Wang, Yi-Xiang Hu, Kun Zhou, Yan Li.

Transcription and replication of the influenza A virus (IAV) genome take place in the nucleus of infected cells, which rely on host factors to aid viral ribonucleoprotein (vRNP) to cross the nuclear pore complex (NPC) and complete the bidirectional nucleocytoplasmic trafficking. Here, we showed that nucleoporin 85 (NUP85), a component of NPC, interacted with RNP subunits polymerase basic 1 (PB1) and polymerase basic 2 (PB2) in an RNA-dependent manner during IAV infection. Knockdown of NUP85 delayed the nuclear import of vRNP, PB1 and PB2, inhibiting polymerase activity and ultimately suppressing viral replication. Further analysis revealed that NUP85 assisted the binding of PB1 to nuclear transport factor Ran-binding protein 5 (RanBP5) and the binding of PB2 to nuclear transport factor importin α1 and importin α7. We also found that NUP85 expression was downregulated upon IAV infection. Together, our study demonstrated that NUP85 positively regulated IAV infection by interacting with viral PB1 and PB2, which may provide new insight into the process of vRNP nuclear import and a novel target for effective antivirals.

Keywords: NUP85; PB1; PB2; RNP; influenza A virus; nuclear import

DOI: https://doi.org/10.3389/fmicb.2022.895779

J Cell Biol. 2022 Sep 05. pii: e202205123. [Epub ahead of print]221(9):

Quality control mechanisms that protect nuclear envelope identity and function.

Philip J Mannino, C Patrick Lusk.

The nuclear envelope (NE) is a specialization of the endoplasmic reticulum with distinct biochemistry that defines inner and outer membranes connected at a pore membrane that houses nuclear pore complexes (NPCs). Quality control mechanisms that maintain the physical integrity and biochemical identity of these membranes are critical to ensure that the NE acts as a selective barrier that also contributes to genome stability and metabolism. As the proteome of the NE is highly integrated, it is challenging to turn over by conventional ubiquitin-proteasome and autophagy mechanisms. Further, removal of entire sections of the NE requires elaborate membrane remodeling that is poorly understood. Nonetheless, recent work has made inroads into discovering specializations of cellular degradative machineries tailored to meeting the unique challenges imposed by the NE. In addition, cells have evolved mechanisms to surveil and repair the NE barrier to protect against the deleterious effects of a breach in NE integrity, in the form of either a ruptured NE or a dysfunctional NPC. Here, we synthesize the most recent work exploring NE quality control mechanisms across eukaryotes.

DOI: https://doi.org/10.1083/jcb.202205123

Mol Biol Cell. 2022 Aug 31. mbcE22050189

A Role for Nup153 in Nuclear Assembly Reveals Differential Requirements for Targeting of Nuclear Envelope Constituents.

Dollie LaJoie, Ayse M Turkmen, Douglas R Mackay, Christopher C Jensen, Vasilisa Aksenova, Maho Niwa, Mary Dasso, Katharine S Ullman.

Assembly of the nucleus following mitosis requires rapid and coordinate recruitment of diverse constituents to the inner nuclear membrane. We have identified an unexpected role for the nucleoporin Nup153 in promoting the continued addition of a subset of nuclear envelope proteins during initial expansion of nascent nuclei. Specifically, disrupting the function of Nup153 interferes with ongoing addition of B-type lamins, lamin B receptor (LBR), and SUN1 early in telophase, after the nuclear envelope (NE) has initially enclosed chromatin. In contrast, effects on lamin A and SUN2 were minimal, pointing to differential requirements for the ongoing targeting of nuclear envelope proteins. Further, distinct mis-targeting phenotypes arose among the proteins that require Nup153 for NE targeting. Thus, disrupting the function of Nup153 in nuclear formation reveals several previously undescribed features important for establishing nuclear architecture: 1) a role for a nuclear basket constituent in ongoing recruitment of nuclear envelope components, 2) two functionally separable phases of nuclear envelope formation in mammalian cells, and 3) distinct requirements of individual nuclear envelope residents for continued targeting during the expansion phase of NE reformation.

DOI: https://doi.org/10.1091/mbc.E22-05-0189

Proc Natl Acad Sci U S A. 2022 Sep 06. 119(36): e2207848119

Key role of exportin 6 in exosome-mediated viral transmission from insect vectors to plants.

Hong Lu, Jiaming Zhu, Jinting Yu, Qiong Li, Lan Luo, Feng Cui.

Exosomes play a key role in virus exocytosis and transmission. The exportin family is usually responsible for cargo nucleocytoplasmic trafficking, and they are frequently found in exosomes. However, the function of exportins sorted in exosomes remains unknown. Here, we successfully isolated "cup holder"-like exosomes from the saliva of ∼30,000 small brown planthoppers, which are vectors of rice stripe virus (RSV). RSV virions were packed in comparatively large exosomes. Four viral genomic RNAs at a certain ratio were identified in the saliva exosomes. The virions contained in the saliva exosomes were capable of replicating and causing disease in rice plants. Interference with each phase of the insect exosome system affected the transmission of RSV from the insect vectors to rice plants. Fragmented exportin 6 was coimmunoprecipitated with viral nucleocapsid protein in saliva and sorted to exosomes via interactions with the cargo sorting protein VPS37a. When the expression of exportin 6 was knocked down, the amounts of RSV secreted in saliva and rice plants were reduced by 60% and 74%, respectively. These results showed that exportin 6 acted as a vehicle for transporting RSV into exosomes to overcome the barrier of insect salivary glands for horizontal transmission. Exportin 6 would represent an ideal target that could be manipulated to control the outbreak of insect-borne viruses in the future.

Keywords: exosome; exportin; insect vector; rice stripe virus; salivary gland

DOI: https://doi.org/10.1073/pnas.2207848119