NNF1 is an essential yeast gene required for proper spindle orientation, nucleolar and nuclear envelope structure and mRNA export

The nuclear envelope is central to nuclear structure and function. It plays a role in maintaining nuclear shape, allowing the exchange of macromolecules between the nucleus and the cytoplasm (via the nuclear pore complexes), and providing attachment sites for microtubules during chromosome segregation and nuclear migration (via the spindle pole body). We have isolated an essential yeast gene, NNF1 that is required for a number of nuclear functions. Cells depleted of Nnf1p or containing a temperature-sensitive nnf1 mutation have elongated microtubules and become bi- and multinucleate. They also have a fragmented nucleolous and accumulate poly(A)+ RNA inside the nucleus. A similar constellation of phenotypes has been reported in cells carrying mutations in a number of nuclear pore proteins, components of the Ran GTPase cycle, and the nuclear localization sequence receptor protein. Our results suggest that Nnf1p plays a role in a number of nuclear functions.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1613916114 ◽

2017 ◽

Vol 114 (11) ◽

pp. E2166-E2175 ◽

Cited By ~ 73

Author(s):

Mingyu Gu ◽

Dollie LaJoie ◽

Opal S. Chen ◽

Alexander von Appen ◽

Mark S. Ladinsky ◽

...

Keyword(s):

Nuclear Envelope ◽

Fission Yeast ◽

Nuclear Membrane ◽

Spindle Pole Body ◽

Spindle Pole ◽

Human Cells ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Loss Of Function

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. InSchizosaccharomyces pombe, deletion of the ATPasevps4leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously inlem2orcmp7, 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.

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Prm3p Is a Pheromone-induced Peripheral Nuclear Envelope Protein Required for Yeast Nuclear Fusion

Molecular Biology of the Cell ◽

10.1091/mbc.e08-10-0987 ◽

2009 ◽

Vol 20 (9) ◽

pp. 2438-2450 ◽

Cited By ~ 21

Author(s):

Shu Shen ◽

Cynthia E. Tobery ◽

Mark D. Rose

Keyword(s):

Nuclear Envelope ◽

Membrane Fusion ◽

Nuclear Membrane ◽

Protein Function ◽

Spindle Pole Body ◽

Point Mutations ◽

Spindle Pole ◽

Nuclear Localization Sequence ◽

Yeast Saccharomyces Cerevisiae ◽

Localization Sequence

Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

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

10.1101/049312 ◽

2016 ◽

Cited By ~ 3

Author(s):

Mingyu Gu ◽

Dollie LaJoie ◽

Opal S. Chen ◽

Alexander Von Appen ◽

Mark S. Ladinsky ◽

...

Keyword(s):

Nuclear Envelope ◽

Fission Yeast ◽

Nuclear Membrane ◽

Spindle Pole Body ◽

Spindle Pole ◽

Human Cells ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Loss Of Function

AbstractESCRT-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. InS. pombe, deletion of the ATPasevps4leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously inlem2orcmp7,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 disc periphery during this window of cell division, and that CHMP7 can bind directly to the C-terminal domain of LEM2in 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.

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The Three Fungal Transmembrane Nuclear Pore Complex Proteins of Aspergillus nidulans Are Dispensable in the Presence of an Intact An-Nup84-120 Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0628 ◽

2009 ◽

Vol 20 (2) ◽

pp. 616-630 ◽

Cited By ~ 73

Author(s):

Hui-Lin Liu ◽

Colin P.C. De Souza ◽

Aysha H. Osmani ◽

Stephen A. Osmani

Keyword(s):

High Temperature ◽

Nuclear Envelope ◽

Aspergillus Nidulans ◽

Nuclear Pore Complex ◽

Mitotic Spindle ◽

Spindle Pole ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Spindle Pole Bodies ◽

Pore Complexes

In Aspergillus nidulans nuclear pore complexes (NPCs) undergo partial mitotic disassembly such that 12 NPC proteins (Nups) form a core structure anchored across the nuclear envelope (NE). To investigate how the NPC core is maintained, we affinity purified the major core An-Nup84-120 complex and identified two new fungal Nups, An-Nup37 and An-ELYS, previously thought to be vertebrate specific. During mitosis the An-Nup84-120 complex locates to the NE and spindle pole bodies but, unlike vertebrate cells, does not concentrate at kinetochores. We find that mutants lacking individual An-Nup84-120 components are sensitive to the membrane destabilizer benzyl alcohol (BA) and high temperature. Although such mutants display no defects in mitotic spindle formation, they undergo mitotic specific disassembly of the NPC core and transient aggregation of the mitotic NE, suggesting the An-Nup84-120 complex might function with membrane. Supporting this, we show cells devoid of all known fungal transmembrane Nups (An-Ndc1, An-Pom152, and An-Pom34) are viable but that An-ndc1 deletion combined with deletion of individual An-Nup84-120 components is either lethal or causes sensitivity to treatments expected to destabilize membrane. Therefore, the An-Nup84-120 complex performs roles, perhaps at the NPC membrane as proposed previously, that become essential without the An-Ndc1 transmembrane Nup.

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The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis

Eukaryotic Cell ◽

10.1128/ec.00231-13 ◽

2013 ◽

Vol 13 (1) ◽

pp. 99-109 ◽

Cited By ~ 11

Author(s):

Meera Govindaraghavan ◽

Alisha A. Lad ◽

Stephen A. Osmani

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Pole Body ◽

Spindle Pole ◽

Cycle Phase ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Content Type ◽

Mitotic Entry ◽

Spindle Pole Bodies ◽

Pore Complexes

ABSTRACTThe G2-M transition inAspergillus nidulansrequires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events.

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nup1 mutants exhibit pleiotropic defects in nuclear pore complex function.

The Journal of Cell Biology ◽

10.1083/jcb.127.2.319 ◽

1994 ◽

Vol 127 (2) ◽

pp. 319-332 ◽

Cited By ~ 45

Author(s):

A M Bogerd ◽

J A Hoffman ◽

D C Amberg ◽

G R Fink ◽

L I Davis

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Structural Integrity ◽

Mutational Analysis ◽

Complex Function ◽

Spindle Pole ◽

Nuclear Pore ◽

Temperature Sensitive ◽

Amino Terminal ◽

Carboxy Terminal

The NUP1 gene of Saccharomyces cerevisiae encodes one member of a family of nuclear pore complex proteins (nucleoporins) conserved from yeast to vertebrates. We have used mutational analysis to investigate the function of Nup1p. Deletion of either the amino- or carboxy-terminal domain confers a lethal phenotype, but partial truncations at either end affect growth to varying extents. Amino-terminal truncation causes mislocalization and degradation of the mutant protein, suggesting that this domain is required for targeting Nup1p to the nuclear pore complex. Carboxy-terminal mutants are stable but do not have wild-type function, and confer a temperature sensitive phenotype. Both import of nuclear proteins and export of poly(A) RNA are defective at the nonpermissive temperature. In addition, nup1 mutant cells become multinucleate at all temperatures, a phenotype suggestive of a defect in nuclear migration. Tubulin staining revealed that the mitotic spindle appears to be oriented randomly with respect to the bud, in spite of the presence of apparently normal cytoplasmic microtubules connecting one spindle pole body to the bud tip. EM analysis showed that the nuclear envelope forms long projections extending into the cytoplasm, which appear to have detached from the bulk of the nucleus. Our results suggest that Nup1p may be required to retain the structural integrity between the nuclear envelope and an underlying nuclear scaffold, and that this connection is required to allow reorientation of the nucleus in response to cytoskeletal forces.

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Expression of TorsinA in a heterologous yeast system reveals interactions with conserved lumenal domains of LINC and nuclear pore complexes

10.1101/421909 ◽

2018 ◽

Author(s):

Madeleine Chalfant ◽

Karl W. Barber ◽

Sapan Borah ◽

David Thaller ◽

C. Patrick Lusk

Keyword(s):

Nuclear Envelope ◽

Genetic Interaction ◽

Spindle Pole ◽

Nuclear Pore ◽

Physical Interaction ◽

Nuclear Pore Complexes ◽

Gene Encoding ◽

Aaa Atpase ◽

Dyt1 Dystonia ◽

Pore Complexes

ABSTRACTDYT1 dystonia is caused by an in-frame deletion of a glutamic acid codon in the gene encoding the AAA+ ATPase TorsinA. TorsinA localizes within the lumen of the nuclear envelope/ER and binds to a membrane-spanning co-factor, LAP1 or LULL1, to form an ATPase; the substrate(s) of TorsinA remain ill defined. Here we use budding yeast, which lack Torsins, to interrogate TorsinA function. We show that TorsinA accumulates at nuclear envelope embedded spindle pole bodies (SPBs) in a way that requires its oligomerization and the conserved SUN-domain protein, Mps3. TorsinA is released from SPBs upon expression of LAP1 and stabilized by LAP1 mutants incapable of stimulating TorsinA ATPase activity, suggesting the recapitulation of a TorsinA-substrate cycle. While the expression of TorsinA or TorsinA-ΔE impacts the fitness of strains expressing mps3 alleles, a genetic interaction with a conserved component of the nuclear pore complex, Pom152, is specific for TorsinA. This specificity is mirrored by a physical interaction between Pom152 and TorsinA, but not TorsinA-ΔE. These data suggest that TorsinA-nucleoporin interactions would be abrogated by TorsinA-ΔE, providing new experimental avenues to interrogate the molecular basis behind nuclear envelope herniations seen in cells lacking TorsinA function.

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Nuclear Pore Complex Number and Distribution throughout theSaccharomyces cerevisiaeCell Cycle by Three-Dimensional Reconstruction from Electron Micrographs of Nuclear Envelopes

Molecular Biology of the Cell ◽

10.1091/mbc.8.11.2119 ◽

1997 ◽

Vol 8 (11) ◽

pp. 2119-2132 ◽

Cited By ~ 147

Author(s):

Mark Winey ◽

Defne Yarar ◽

Thomas H. Giddings ◽

David N. Mastronarde

Keyword(s):

Cell Cycle ◽

Nuclear Envelope ◽

Electron Micrographs ◽

Three Dimensional ◽

Spindle Pole ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nuclear Surface ◽

Yeast Saccharomyces Cerevisiae ◽

Cell Cycle Stage

The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells. Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs. Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined. NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G1-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle. However, the accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells. Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance. However, NPCs were found in large clusters over regions of the nuclear envelope. Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.

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Members of the RSC Chromatin-Remodeling Complex Are Required for Maintaining Proper Nuclear Envelope Structure and Pore Complex Localization

Molecular Biology of the Cell ◽

10.1091/mbc.e09-07-0615 ◽

2010 ◽

Vol 21 (6) ◽

pp. 1072-1087 ◽

Cited By ~ 33

Author(s):

Laura C. Titus ◽

T. Renee Dawson ◽

Deborah J. Rexer ◽

Kathryn J. Ryan ◽

Susan R. Wente

Keyword(s):

Nuclear Envelope ◽

Chromatin Remodeling ◽

Fluorescent Protein ◽

Nuclear Periphery ◽

Structural Defects ◽

Nuclear Pore ◽

Temperature Sensitive ◽

Nuclear Pore Complexes ◽

Chromatin Remodeling Complex ◽

Green Fluorescent

The assembly, distribution, and functional integrity of nuclear pore complexes (NPCs) in the nuclear envelope (NE) are key determinants in the nuclear periphery architecture. However, the mechanisms controlling proper NPC and NE structure are not fully defined. We used two different genetic screening approaches to identify Saccharomyces cerevisiae mutants with defects in NPC localization. The first approach examined green fluorescent protein (GFP)-Nic96 in 531 strains from the yeast Tet-promoters Hughes Collection with individual essential genes expressed from a doxycycline-regulated promoter (TetO7-orf). Under repressive conditions, depletion of the protein encoded by 44 TetO7-orf strains resulted in mislocalized GFP-Nic96. These included STH1, RSC4, RSC8, RSC9, RSC58, ARP7, and ARP9, each encoding components of the RSC chromatin remodeling complex. Second, a temperature-sensitive sth1-F793S (npa18-1) mutant was identified in an independent genetic screen for NPC assembly (npa) mutants. NPC mislocalization in the RSC mutants required new protein synthesis and ongoing transcription, confirming that lack of global transcription did not underlie the phenotypes. Electron microscopy studies showed significantly altered NEs and nuclear morphology, with coincident cytoplasmic membrane sheet accumulation. Strikingly, increasing membrane fluidity with benzyl alcohol treatment prevented the sth1-F793S NE structural defects and NPC mislocalization. We speculate that NE structure is functionally linked to proper chromatin architecture.

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