scholarly journals Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

2004 ◽  
Vol 167 (6) ◽  
pp. 1051-1062 ◽  
Author(s):  
Tomoyuki Ohba ◽  
Eric C. Schirmer ◽  
Takeharu Nishimoto ◽  
Larry Gerace
Keyword(s):  
Nuclear Membrane ◽  
Lateral Diffusion ◽  
Nuclear Lamina ◽  
Atp Depletion ◽  
Nuclear Pore ◽  
Temperature Dependent ◽  
Inner Nuclear Membrane ◽  
Dependent Transport ◽  
Integral Proteins ◽  
Reduced Temperature

Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

scholarly journals Internuclear exchange of an inner nuclear membrane protein (p55) in heterokaryons: in vivo evidence for the interaction of p55 with the nuclear lamina.

1990 ◽  
Vol 111 (6) ◽  
pp. 2225-2234 ◽  
Author(s):  
L Powell ◽  
B Burke
Keyword(s):  
Nuclear Membrane ◽  
Lateral Diffusion ◽  
Nuclear Lamina ◽  
Membrane System ◽  
Mouse Cell ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Inner Nuclear Membrane ◽  
Pore Complexes

The movement between nuclei of an integral protein of the inner nuclear membrane has been studied in rat/mouse and rat/hamster heterokaryons. This protein, p55, was found to equilibrate between nuclei over a period of approximately 6 h in the absence of new protein synthesis. When rat/mouse heterokaryons were constructed using an undifferentiated murine embryonal carcinoma (P19), which lacks lamins A and C, no accumulation of p55 in the mouse cell nucleus was observed. However, P19 nuclei could be rendered competent to accumulate p55 by transfecting the parent cells with human lamin A before cell fusion, supporting the notion that p55 may interact with the nuclear lamina. Since p55 does not appear to be able to dissociate from the nuclear membrane, it is concluded that this exchange between nuclei does not occur in the aqueous phase and instead is probably membrane mediated. It is proposed that this protein may be free to move between the inner and outer nuclear membranes via the continuities at the nuclear pore complexes and that transfer between nuclei occurs via lateral diffusion through the peripheral ER, which appears to form a single continuous membrane system in these heterokaryons. One implication of these observations is that accumulation of at least some integral proteins in the inner nuclear membrane may be mediated by interactions with other nuclear components and may not require a single defined targeting sequence.

scholarly journals Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina.

1988 ◽  
Vol 107 (6) ◽  
pp. 2029-2036 ◽  
Author(s):  
A Senior ◽  
L Gerace
Keyword(s):  
Membrane Proteins ◽  
Nuclear Membrane ◽  
Immunofluorescence Microscopy ◽  
Nuclear Lamina ◽  
Integral Membrane Proteins ◽  
Inner Nuclear Membrane ◽  
Attachment Sites ◽  
Nonionic Detergent ◽  
Pore Complexes ◽  
Integral Proteins

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.

Sequential PKC- and Cdc2-mediated phosphorylation events elicit zebrafish nuclear envelope disassembly

1999 ◽  
Vol 112 (6) ◽  
pp. 977-987 ◽  
Author(s):  
P. Collas
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Cdc2 Kinase ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Nuclear Envelope Breakdown ◽  
Simultaneous Inhibition ◽  
Integral Proteins

Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis.

scholarly journals Inner nuclear membrane protein transport is mediated by multiple mechanisms

2008 ◽  
Vol 36 (6) ◽  
pp. 1373-1377 ◽  
Author(s):  
Nikolaj Zuleger ◽  
Nadia Korfali ◽  
Eric C. Schirmer
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Protein Transport ◽  
Protein Translocation ◽  
Lateral Diffusion ◽  
Membrane System ◽  
Nuclear Pore ◽  
Inner Nuclear Membrane ◽  
Classical Transport

Work in the nuclear transport field has led to an incredibly detailed description of protein translocation through the central channel of the nuclear pore complex, yet the mechanism by which nuclear envelope transmembrane proteins reach the inner nuclear membrane after synthesis in the endoplasmic reticulum is still hotly debated. Three different translocation models have gained experimental support: (i) simple lateral diffusion through the nuclear envelope membrane system; (ii) translocation by vesicle fusion events; and (iii) a variation on classical transport mediated by the nuclear pore complex. Although these models appear to be mutually exclusive, in the present paper we argue that they probably all function for different inner nuclear membrane proteins according to their unique characteristics.

scholarly journals Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

2015 ◽  
Vol 26 (18) ◽  
pp. 3301-3312 ◽  
Author(s):  
Annemarie Kralt ◽  
Noorjahan B. Jagalur ◽  
Vincent van den Boom ◽  
Ravi K. Lokareddy ◽  
Anton Steen ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Nuclear Membrane ◽  
Nuclear Accumulation ◽  
Small Subset ◽  
Nuclear Pore ◽  
Simple Diffusion ◽  
Inner Nuclear Membrane ◽  
Intrinsically Disordered ◽  
Localization Signal ◽  
Dependent Transport

Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins.

scholarly journals Synthesis, transport and incorporation into the nuclear envelope of A-type lamins and inner nuclear membrane proteins

2011 ◽  
Vol 39 (6) ◽  
pp. 1758-1763 ◽  
Author(s):  
Jose M. González ◽  
Vicente Andrés
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Complex Structure ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Inner Nuclear Membrane ◽  
Perinuclear Space ◽  
Interphase Cells ◽  
Pore Complexes

The mammalian NE (nuclear envelope), which separates the nucleus from the cytoplasm, is a complex structure composed of nuclear pore complexes, the outer and inner nuclear membranes, the perinuclear space and the nuclear lamina (A- and B-type lamins). The NE is completely disassembled and reassembled at each cell division. In the present paper, we review recent advances in the understanding of the mechanisms implicated in the transport of inner nuclear membrane and nuclear lamina proteins from the endoplasmic reticulum to the nucleus in interphase cells and mitosis, with special attention to A-type lamins.

Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

2020 ◽  
Vol 477 (14) ◽  
pp. 2715-2720
Author(s):  
Susana Castro-Obregón
Keyword(s):  
Cellular Senescence ◽  
Nuclear Membrane ◽  
Chromatin Structure ◽  
Cholesterol Synthesis ◽  
Reductase Activity ◽  
Chimeric Protein ◽  
Nuclear Lamina ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Lamin B Receptor

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

Function and assembly of nuclear pore complex proteins

1999 ◽  
Vol 77 (4) ◽  
pp. 321-329 ◽  
Author(s):  
Khaldon Bodoor ◽  
Sarah Shaikh ◽  
Paul Enarson ◽  
Sharmin Chowdhury ◽  
Davide Salina ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Current View ◽  
Nuclear Pore ◽  
Dynamic Component ◽  
Inner Nuclear Membrane ◽  
Inner Nuclear Membrane Protein ◽  
Nuclear Membrane Protein

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. The current view of NPC organization features a massive symmetrical framework that is embedded in the double membranes of the nuclear envelope. It embraces a central channel of as yet ill-defined structure but which may accommodate particles with diameters up to 26 nm provided that they bear specific import/export signals. Attached to both faces of the central framework are peripheral structures, short cytoplasmic filaments, and a nuclear basket assembly, which interact with molecules transiting the NPC. The mechanisms of assembly and the nature of NPC structural intermediates are still poorly understood. However, mutagenesis and expression studies have revealed discrete sequences within certain NPC proteins that are necessary and sufficient for their appropriate targeting. In addition, some details are emerging from observations on cells undergoing mitosis where 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 to form nuclear envelopes in the two daughter cells. To date, it has been possible to define a time course of postmitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral inner nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a dynamic component of the nuclear basket, associates with chromatin towards the end of anaphase coincident with, although independent of, the inner nuclear membrane protein, LAP2. 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, p54, p45) 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 and which may therefore represent an essential component of the central framework of the NPC. Key words: nuclear pore complex, nucleoporin, mitosis, nuclear transport

scholarly journals Nuclear envelope organization in Dictyostelium discoideum

2019 ◽  
Vol 63 (8-9-10) ◽  
pp. 509-519 ◽  
Author(s):  
Petros Batsios ◽  
Ralph Gräf ◽  
Michael P. Koonce ◽  
Denis A. Larochelle ◽  
Irene Meyer
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Major Constituent ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Linc Complex ◽  
Family Protein ◽  
Import And Export ◽  
Pore Complexes

The nuclear envelope consists of the outer and the inner nuclear membrane, the nuclear lamina and the nuclear pore complexes, which regulate nuclear import and export. The major constituent of the nuclear lamina of Dictyostelium is the lamin NE81. It can form filaments like B-type lamins and it interacts with Sun1, as well as with the LEM/HeH-family protein Src1. Sun1 and Src1 are nuclear envelope transmembrane proteins involved in the centrosome-nucleus connection and nuclear envelope stability at the nucleolar regions, respectively. In conjunction with a KASH-domain protein, Sun1 usually forms a so-called LINC complex. Two proteins with functions reminiscent of KASH-domain proteins at the outer nuclear membrane of Dictyostelium are known; interaptin which serves as an actin connector and the kinesin Kif9 which plays a role in the microtubule-centrosome connector. However, both of these lack the conserved KASH-domain. The link of the centrosome to the nuclear envelope is essential for the insertion of the centrosome into the nuclear envelope and the appropriate spindle formation. Moreover, centrosome insertion is involved in permeabilization of the mitotic nucleus, which ensures access of tubulin dimers and spindle assembly factors. Our recent progress in identifying key molecular players at the nuclear envelope of Dictyostelium promises further insights into the mechanisms of nuclear envelope dynamics.

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