scholarly journals The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes

2007 ◽  
Vol 178 (5) ◽  
pp. 799-812 ◽  
Author(s):  
John J. Scarcelli ◽  
Christine A. Hodge ◽  
Charles N. Cole
Keyword(s):  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Membrane Dynamics ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cold Sensitive ◽  
Low Levels ◽  
Lower Temperature ◽  
Pore Complexes ◽  
And Function

Although the structure and function of components of the nuclear pore complex (NPC) have been the focus of many studies, relatively little is known about NPC biogenesis. In this study, we report that Apq12 is required for efficient NPC biogenesis in Saccharomyces cerevisiae. Apq12 is an integral membrane protein of the nuclear envelope (NE) and endoplasmic reticulum. Cells lacking Apq12 are cold sensitive for growth, and a subset of their nucleoporins (Nups), those that are primarily components of the cytoplasmic fibrils of the NPC, mislocalize to the cytoplasm. APQ12 deletion also causes defects in NE morphology. In the absence of Apq12, most NPCs appear to be associated with the inner but not the outer nuclear membrane. Low levels of benzyl alcohol, which increases membrane fluidity, prevented Nup mislocalization and restored the proper localization of Nups that had accumulated in cytoplasmic foci upon a shift to lower temperature. Thus, Apq12p connects nuclear pore biogenesis to the dynamics of the NE.

scholarly journals Nucleus–Vacuole Junctions in Saccharomyces cerevisiae Are Formed Through the Direct Interaction of Vac8p with Nvj1p

2000 ◽  
Vol 11 (7) ◽  
pp. 2445-2457 ◽  
Author(s):  
Xiaozhou Pan ◽  
Paul Roberts ◽  
Yan Chen ◽  
Erik Kvam ◽  
Natalyia Shulga ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Fluorescent Protein ◽  
Close Contact ◽  
Direct Interaction ◽  
Integral Membrane Protein ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Importin Α ◽  
Green Fluorescent ◽  
Pore Complexes

Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including β-catenin and importin α, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus–vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40–60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in “orphan” rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Δ cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Δ andvac8-Δ cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.

scholarly journals The SUN protein Mps3 controls Ndc1 distribution and function on the nuclear membrane

2014 ◽  
Vol 204 (4) ◽  
pp. 523-539 ◽  
Author(s):  
Jingjing Chen ◽  
Christine J. Smoyer ◽  
Brian D. Slaughter ◽  
Jay R. Unruh ◽  
Sue L. Jaspersen
Keyword(s):  
Spindle Pole Body ◽  
Integral Membrane Protein ◽  
Spindle Pole ◽  
Correlation Spectroscopy ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
The Sun ◽  
Pore Complexes ◽  
And Function

In closed mitotic systems such as Saccharomyces cerevisiae, nuclear pore complexes (NPCs) and the spindle pole body (SPB) must assemble into an intact nuclear envelope (NE). Ndc1 is a highly conserved integral membrane protein involved in insertion of both complexes. In this study, we show that Ndc1 interacts with the SUN domain–containing protein Mps3 on the NE in live yeast cells using fluorescence cross-correlation spectroscopy. Genetic and molecular analysis of a series of new ndc1 alleles allowed us to understand the role of Ndc1–Mps3 binding at the NE. We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication. Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component. Based on our data we propose that the Ndc1–Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB.

scholarly journals Concentration-dependent lamin assembly and its roles in the localization of other nuclear proteins

2014 ◽  
Vol 25 (8) ◽  
pp. 1287-1297 ◽  
Author(s):  
Yuxuan Guo ◽  
Youngjo Kim ◽  
Takeshi Shimi ◽  
Robert D. Goldman ◽  
Yixian Zheng
Keyword(s):  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Lamin A ◽  
Nuclear Pore Complexes ◽  
Developmental Defects ◽  
Protein Levels ◽  
Lamin B1 ◽  
Mouse Cells ◽  
Pore Complexes ◽  
And Function

The nuclear lamina (NL) consists of lamin polymers and proteins that bind to the polymers. Disruption of NL proteins such as lamin and emerin leads to developmental defects and human diseases. However, the expression of multiple lamins, including lamin-A/C, lamin-B1, and lamin-B2, in mammals has made it difficult to study the assembly and function of the NL. Consequently, it has been unclear whether different lamins depend on one another for proper NL assembly and which NL functions are shared by all lamins or are specific to one lamin. Using mouse cells deleted of all or different combinations of lamins, we demonstrate that the assembly of each lamin into the NL depends primarily on the lamin concentration present in the nucleus. When expressed at sufficiently high levels, each lamin alone can assemble into an evenly organized NL, which is in turn sufficient to ensure the even distribution of the nuclear pore complexes. By contrast, only lamin-A can ensure the localization of emerin within the NL. Thus, when investigating the role of the NL in development and disease, it is critical to determine the protein levels of relevant lamins and the intricate shared or specific lamin functions in the tissue of interest.

scholarly journals ESCRT recruitment by the S. cerevisiae inner nuclear membrane protein Heh1 is regulated by Hub1-mediated alternative splicing

2020 ◽  
Vol 133 (24) ◽  
pp. jcs250688 ◽  
Author(s):  
Matías Capella ◽  
Lucía Martín Caballero ◽  
Boris Pfander ◽  
Sigurd Braun ◽  
Stefan Jentsch
Keyword(s):  
Alternative Splicing ◽  
Membrane Protein ◽  
Nuclear Membrane ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Inner Nuclear Membrane ◽  
Growth Defects ◽  
Pore Complexes ◽  
Inner Nuclear Membrane Protein ◽  
Nuclear Membrane Protein

ABSTRACTMisassembled nuclear pore complexes (NPCs) are removed by sealing off the surrounding nuclear envelope (NE), which is conducted by the endosomal sorting complexes required for transport (ESCRT) machinery. Recruitment of ESCRT proteins to the NE is mediated by the interaction between the ESCRT member Chm7 and the inner nuclear membrane protein Heh1, which belongs to the conserved LEM family. Increased ESCRT recruitment results in excessive membrane scission at damage sites but its regulation remains poorly understood. Here, we show that Hub1-mediated alternative splicing of HEH1 pre-mRNA, resulting in production of its shorter form Heh1-S, is critical for the integrity of the NE in Saccharomyces cerevisiae. ESCRT-III mutants lacking Hub1 or Heh1-S display severe growth defects and accumulate improperly assembled NPCs. This depends on the interaction of Chm7 with the conserved MSC domain, which is only present in the longer variant Heh1-L. Heh1 variants assemble into heterodimers, and we demonstrate that a unique splice segment in Heh1-S suppresses growth defects associated with the uncontrolled interaction between Heh1-L and Chm7. Together, our findings reveal that Hub1-mediated splicing generates Heh1-S to regulate ESCRT recruitment to the NE.This article has an associated First Person interview with the first author of the paper.

scholarly journals A role for Gle1, a regulator of DEAD-box RNA helicases, at centrosomes and basal bodies

2017 ◽  
Vol 28 (1) ◽  
pp. 120-127 ◽  
Author(s):  
Li-En Jao ◽  
Abdalla Akef ◽  
Susan R. Wente
Keyword(s):  
Mrna Export ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Rna Helicases ◽  
Microtubule Organization ◽  
Developmental Defects ◽  
Dead Box ◽  
Pericentriolar Material ◽  
Pore Complexes ◽  
And Function

Control of organellar assembly and function is critical to eukaryotic homeostasis and survival. Gle1 is a highly conserved regulator of RNA-dependent DEAD-box ATPase proteins, with critical roles in both mRNA export and translation. In addition to its well-defined interaction with nuclear pore complexes, here we find that Gle1 is enriched at the centrosome and basal body. Gle1 assembles into the toroid-shaped pericentriolar material around the mother centriole. Reduced Gle1 levels are correlated with decreased pericentrin localization at the centrosome and microtubule organization defects. Of importance, these alterations in centrosome integrity do not result from loss of mRNA export. Examination of the Kupffer’s vesicle in Gle1-depleted zebrafish revealed compromised ciliary beating and developmental defects. We propose that Gle1 assembly into the pericentriolar material positions the DEAD-box protein regulator to function in localized mRNA metabolism required for proper centrosome function.

scholarly journals Targeting and function in mRNA export of nuclear pore complex protein Nup153.

1996 ◽  
Vol 134 (5) ◽  
pp. 1141-1156 ◽  
Author(s):  
R Bastos ◽  
A Lin ◽  
M Enarson ◽  
B Burke
Keyword(s):  
Nuclear Pore Complex ◽  
Protein Import ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Terminal Domain ◽  
General Decline ◽  
Complex Protein ◽  
Pore Complexes ◽  
And Function

Nup153 is a large (153 kD) O-linked glyco-protein which is a component of the basket structure located on the nucleoplasmic face of nuclear pore complexes. This protein exhibits a tripartite structure consisting of a zinc finger domain flanked by large (60-70 kD) NH2- and COOH-terminal domains. When full-length human Nup153 is expressed in BHK cells, it accumulates appropriately at the nucleoplasmic face of the nuclear envelope. Targeting information for Nup153 resides in the NH2-terminal domain since this region of the molecule can direct an ordinarily cytoplasmic protein, pyruvate kinase, to the nuclear face of the nuclear pore complex. Overexpression of Nup153 results in the dramatic accumulation of nuclear poly (A)+ RNA, suggesting an inhibition of RNA export from the nucleus. This is not due to a general decline in nucleocytoplasmic transport or to occlusion or loss of nuclear pore complexes since nuclear protein import is unaffected. While overexpression of certain Nup153 constructs was found to result in the formation of unusual intranuclear membrane arrays, this structural phenotype could not be correlated with the effects on poly (A)+ RNA distribution. The RNA trafficking defect was, however, dependent upon the Nup153 COOH-terminal domain which contains most of the XFXFG repeats. It is proposed that this region of Nup153, lying within the distal ring of the nuclear basket, represents a docking site for mRNA molecules exiting the nucleus.

scholarly journals Dunking into the Lipid Bilayer: How Direct Membrane Binding of Nucleoporins Can Contribute to Nuclear Pore Complex Structure and Assembly

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3601
Author(s):  
Mohamed Hamed ◽  
Wolfram Antonin
Keyword(s):  
Nuclear Envelope ◽  
Current Knowledge ◽  
Complex Structure ◽  
Transmembrane Proteins ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Membrane Spanning ◽  
Pore Complexes ◽  
And Function ◽  
Membrane Spanning Domains

Nuclear pore complexes (NPCs) mediate the selective and highly efficient transport between the cytoplasm and the nucleus. They are embedded in the two membrane structure of the nuclear envelope at sites where these two membranes are fused to pores. A few transmembrane proteins are an integral part of NPCs and thought to anchor these complexes in the nuclear envelope. In addition, a number of nucleoporins without membrane spanning domains interact with the pore membrane. Here we review our current knowledge of how these proteins interact with the membrane and how this interaction can contribute to NPC assembly, stability and function as well as shaping of the pore membrane.

Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis

1999 ◽  
Vol 112 (13) ◽  
pp. 2253-2264 ◽  
Author(s):  
K. Bodoor ◽  
S. Shaikh ◽  
D. Salina ◽  
W.H. Raharjo ◽  
R. Bastos ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Time Course ◽  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Confocal Immunofluorescence Microscopy ◽  
Pore Complexes ◽  
Nuclear Membrane Protein

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. With a mass of about 125 MDa, NPCs are thought to be composed of 50 or more distinct protein subunits, each present in multiple copies. During mitosis in higher cells the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized. Using both conventional and digital confocal immunofluorescence microscopy we have been able to define a time course of post-mitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a component of the nuclear basket, associates with chromatin towards the end of anaphase, in parallel with the inner nuclear membrane protein, LAP2. However, immunogold labeling suggests that the initial Nup153 chromatin association is membrane-independent. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, 54, 45) during mitosis and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates.

Cryo - Fesem of Subcellular Organisation Using an In-Lens Rod with A Vacuum Transfer System

2001 ◽  
Vol 7 (S2) ◽  
pp. 724-725
Author(s):  
Terry Allen ◽  
Sandra Rutherford ◽  
Steve Bagley ◽  
Bob Morrison ◽  
Martin Goldberg
Keyword(s):  
High Resolution ◽  
Structural Information ◽  
Membrane Dynamics ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Surface Imaging ◽  
Thin Sections ◽  
Critical Point Drying ◽  
Cryo Preservation ◽  
Pore Complexes

The recent increase in use of Field Emission Scanning Instruments in biology has shown the potential of these systems for sub-cellular imaging, as they essentially provide transmission EM levels of resolution for surface imaging. in structures lacking inherent contrast in thin sections, such as nuclear pore complexes,(NPC) high resolution surface imaging has provided significant complementary structural information to the macromolecular architecture of the NPC. (Goldberg and Allen, 1996). A series of intermediate structural forms of the NPC during its formation has also been described using FESEM, which would not have been resolvable in TEM.(Goldberg, Weise, Allen, Wilson, 1997). All this work was performed with chemical fixation, dehydration and critical point drying, followed by high resolution Chromium coating. We now are extending these studies to a cryo-preservation approach, mindful of the requirement of rapid freezing to preserve the more labile events of membrane dynamics, and also to circumvent the requirement to expose membranes to organic solvents during conventional dehydration.

scholarly journals Nup358 integrates nuclear envelope breakdown with kinetochore assembly

2003 ◽  
Vol 162 (6) ◽  
pp. 991-1001 ◽  
Author(s):  
Davide Salina ◽  
Paul Enarson ◽  
J.B. Rattner ◽  
Brian Burke
Keyword(s):  
Nuclear Envelope ◽  
Mitotic Checkpoint ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Envelope Breakdown ◽  
Chromosome Congression ◽  
Chromatid Segregation ◽  
Pore Complexes ◽  
Membrane Components ◽  
And Function

Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.

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