Intracellular trafficking of MAN1, an integral protein of the nuclear envelope inner membrane

2002 ◽  
Vol 115 (7) ◽  
pp. 1361-1371 ◽  
Author(s):  
Wei Wu ◽  
Feng Lin ◽  
Howard J. Worman
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Subcellular Location ◽  
Globular Domain ◽  
Retention Model ◽  
Inner Nuclear Membrane ◽  
Terminal Domain ◽  
Integral Protein ◽  
Green Fluorescent

MAN1 is an integral protein of the inner nuclear membrane that shares the LEM domain, a conserved globular domain of approximately 40 amino acids, with lamina-associated polypeptide (LAP) 2 and emerin. Confocal immuofluorescence microscopy studies of the intracellular targeting of truncated forms of MAN1 showed that the nucleoplasmic, N-terminal domain is necessary for inner nuclear membrane retention. A protein containing the N-terminal domain with the first transmembrane segment of MAN1 is retained in the inner nuclear membrane, whereas the transmembrane segments with the C-terminal domain of MAN1 is not targeted to the inner nuclear membrane. The N-terminal domain of MAN1 is also sufficient for inner nuclear membrane targeting as it can target a chimeric type II integral protein to this subcellular location. Deletion mutants of the N-terminal of MAN1 are not efficiently retained in the inner nuclear membrane. When the N-terminal domain of MAN1 is increased in size from∼50 kDa to ∼100 kDa, the protein cannot reach the inner nuclear membrane. Fluorescence recovery after photobleaching experiments of MAN1 fused to green fluorescent protein show that the fusion protein is relatively immobile in the nuclear envelope compared with the endoplasmic reticulum of interphase cells, suggesting binding to a nuclear component. These results are in agreement with the `diffusion-retention' model for targeting integral proteins to the inner nuclear membrane.

scholarly journals Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane.

1995 ◽  
Vol 130 (1) ◽  
pp. 15-27 ◽  
Author(s):  
B Soullam ◽  
H J Worman
Keyword(s):  
Membrane Protein ◽  
Nuclear Membrane ◽  
Structural Features ◽  
Inner Nuclear Membrane ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Integral Protein ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Amino Terminal Domain

We have examined transfected cells by immunofluorescence microscopy to determine the signals and structural features required for the targeting of integral membrane proteins to the inner nuclear membrane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nucleoplasmic, amino-terminal domain and a carboxyl-terminal domain with eight putative transmembrane segments. The amino-terminal domain of LBR can target both a cytosolic protein to the nucleus and a type II integral protein to the inner nuclear membrane. Neither a nuclear localization signal (NLS) of a soluble protein, nor full-length histone H1, can target an integral protein to the inner nuclear membrane although they can target cytosolic proteins to the nucleus. The addition of an NLS to a protein normally located in the inner nuclear membrane, however, does not inhibit its targeting. When the amino-terminal domain of LBR is increased in size from approximately 22.5 to approximately 70 kD, the chimeric protein cannot reach the inner nuclear membrane. The carboxyl-terminal domain of LBR, separated from the amino-terminal domain, also concentrates in the inner nuclear membrane, demonstrating two nonoverlapping targeting signals in this protein. Signals and structural features required for the inner nuclear membrane targeting of proteins are distinct from those involved in targeting soluble polypeptides to the nucleoplasm. The structure of the nucleocytoplasmic domain of an inner nuclear membrane protein also influences targeting, possibly because of size constraints dictated by the lateral channels of the nuclear pore complexes.

Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein

1999 ◽  
Vol 112 (11) ◽  
pp. 1709-1719 ◽  
Author(s):  
C. Ostlund ◽  
J. Ellenberg ◽  
E. Hallberg ◽  
J. Lippincott-Schwartz ◽  
H.J. Worman
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Muscular Dystrophy ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Transmembrane Segment ◽  
Inner Nuclear Membrane ◽  
Amino Terminal ◽  
Green Fluorescent

Emerin is an integral protein of the inner nuclear membrane that is mutated or not expressed in patients with Emery-Dreifuss muscular dystrophy. Confocal immunofluorescence microscopy studies of the intracellular targeting of truncated forms of emerin, some of which are found in patients with Emery-Dreifuss muscular dystrophy, show that the nucleoplasmic, amino-terminal domain is necessary and sufficient for nuclear retention. When this domain is fused to a transmembrane segment of an integral membrane protein of the ER/plasma membrane, the chimeric protein is localized in the inner nuclear membrane. The transmembrane segment of emerin is not targeted to the inner nuclear membrane. Fluorescence photobleaching experiments of emerin fused to green fluorescent protein demonstrate that the diffusional mobility (D) of emerin is decreased in the inner nuclear membrane (D=0.10+/-0.01 microm2/second) compared to the ER membrane (D=0.32+/-0.01 microm2/second). This is in agreement with a model where integral proteins reach the inner nuclear membrane by lateral diffusion and are retained there by association with nucleoplasmic components. Some overexpressed emerin-green fluorescent protein also reaches the plasma membrane of transfected cells, where its diffusion is similar to that in the inner nuclear membrane, suggesting that emerin may also associate with non-nuclear structures.

scholarly journals Association of Prenylated Proteins with the Plasma Membrane and the Inner Nuclear Membrane Is Mediated by the Same Membrane-targeting Motifs

2000 ◽  
Vol 11 (9) ◽  
pp. 3233-3246 ◽  
Author(s):  
Helmut Hofemeister ◽  
Klaus Weber ◽  
Reimer Stick
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Membrane Association ◽  
Membrane Targeting ◽  
Inner Nuclear Membrane ◽  
Nuclear Lamins ◽  
Green Fluorescent ◽  
Caax Motif

Targeting of nuclear lamins to the inner nuclear envelope membrane requires a nuclear localization signal and CaaX motif–dependent posttranslational modifications, including isoprenylation and carboxyl methylation. These modifications, although necessary for membrane targeting, are not sufficient to mediate stable association with membranes. We show that two variants of lamin B3 (i.e., B3a and B3b) exist in Xenopus oocytes. They are encoded by two alternatively spliced, developmentally regulated mRNAs. The two lamin variants differ greatly in their membrane association in meiotically matured eggs. The presence of an extra cysteine residue (as a potential palmitoylation site) and a basic cluster in conjunction with the CaaX motif function as secondary targeting signals responsible for the stable membrane association of lamin B3b in Xenopuseggs. Moreover, transfection experiments with Green Fluorescent Protein lamin tail chimeras and with a Green Fluorescent Protein N-Ras chimera show that these secondary motifs are sufficient to target proteins to the inner nuclear membrane and/or the plasma membrane. Implications for the intracellular trafficking of doubly lipidated proteins are discussed.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

scholarly journals The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

2001 ◽  
Vol 152 (2) ◽  
pp. 385-400 ◽  
Author(s):  
Patrick Heun ◽  
Thierry Laroche ◽  
M.K. Raghuraman ◽  
Susan M. Gasser
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Yeast Cells ◽  
G1 Phase ◽  
Nuclear Pore ◽  
Origin Firing ◽  
Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

scholarly journals Unrestrained ESCRT-III drives chromosome fragmentation and micronuclear catastrophe

2019 ◽  
Author(s):  
Marina Vietri ◽  
Sebastian W. Schultz ◽  
Aurélie Bellanger ◽  
Carl M. Jones ◽  
Camilla Raiborg ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Genome Stability ◽  
Membrane Deformation ◽  
Chromosome Fragmentation ◽  
Inner Nuclear Membrane ◽  
Membrane Rupture ◽  
Membrane Fission ◽  
Stable Association ◽  
Inner Nuclear Membrane Protein

AbstractThe ESCRT-III membrane fission machinery1,2 restores nuclear envelope integrity during mitotic exit3,4 and interphase5,6. Whereas primary nuclei resealing takes minutes, micronuclear envelope ruptures appear irreversible and result in catastrophic collapse associated with chromosome fragmentation and rearrangements (chromothripsis), thought to be a major driving force in cancer development7-10. Despite its importance11-13, the mechanistic underpinnings of nuclear envelope sealing in primary nuclei and the defects observed in micronuclei remain largely unknown. Here we show that CHMP7, the nucleator of ESCRT-III filaments at the nuclear envelope3,14, and the inner nuclear membrane protein LEMD215 act as a compartmentalization sensor detecting the loss of nuclear integrity. In cells with intact nuclear envelope, CHMP7 is actively excluded from the nucleus to preclude its binding to LEMD2. Nuclear influx of CHMP7 results in stable association with LEMD2 at the inner nuclear membrane that licenses local polymerization of ESCRT-III. Tight control of nuclear CHMP7 levels is critical, as induction of nuclear CHMP7 mutants is sufficient to induce unrestrained growth of ESCRT-III foci at the nuclear envelope, causing dramatic membrane deformation, local DNA torsional stress, single-stranded DNA formation and fragmentation of the underlying chromosomes. At micronuclei, membrane rupture is not associated with repair despite timely recruitment of ESCRT-III. Instead, micronuclei inherently lack the capacity to restrict accumulation of CHMP7 and LEMD2. This drives unrestrained ESCRT-III recruitment, membrane deformation and DNA defects that strikingly resemble those at primary nuclei upon induction of nuclear CHMP7 mutants. Preventing ESCRT-III recruitment suppresses membrane deformation and DNA damage, without restoring nucleocytoplasmic compartmentalization. We propose that the ESCRT-III nuclear integrity surveillance machinery is a double-edged sword, as its exquisite sensitivity ensures rapid repair at primary nuclei while causing unrestrained polymerization at micronuclei, with catastrophic consequences for genome stability16-18.

scholarly journals Green Fluorescent Chimeras Indicate Nonpolar Localization of Pullulanase Secreton Components PulL and PulM

2006 ◽  
Vol 188 (8) ◽  
pp. 2928-2935 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Olivera Francetic ◽  
Anthony P. Pugsley
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Klebsiella Oxytoca ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Native Proteins ◽  
Type Ii Secretion System ◽  
Green Fluorescent

ABSTRACT The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.

A novel linker histone-like protein is associated with cytoplasmic filaments inCaenorhabditis elegans

2002 ◽  
Vol 115 (14) ◽  
pp. 2881-2891
Author(s):  
Monika A. Jedrusik ◽  
Stefan Vogt ◽  
Peter Claus ◽  
Ekkehard Schulze
Keyword(s):  
Rna Interference ◽  
Fluorescent Protein ◽  
Muscle Growth ◽  
Egg Laying ◽  
Globular Domain ◽  
Protein Fusion ◽  
Linker Histones ◽  
C Elegans ◽  
Fusion Protein Expression ◽  
Green Fluorescent

The histone H1 complement of Caenorhabditis elegans contains a single unusual protein, H1.X. Although H1.X possesses the globular domain and the canonical three-domain structure of linker histones, the amino acid composition of H1.X is distinctly different from conventional linker histones in both terminal domains. We have characterized H1.X in C. elegans by antibody labeling, green fluorescent protein fusion protein expression and RNA interference. Unlike normal linker histones, H1.X is a cytoplasmic as well as a nuclear protein and is not associated with chromosomes. H1.X is most prominently expressed in the marginal cells of the pharynx and is associated with a peculiar cytoplasmic cytoskeletal structure therein, the tonofilaments. Additionally H1.X::GFP is expressed in the cytoplasm of body and vulva muscle cells, neurons, excretory cells and in the nucleoli of embryonic blastomeres and adult gut cells. RNA interference with H1.X results in uncoordinated and egg laying defective animals, as well as in a longitudinally enlarged pharynx. These phenotypes indicate a cytoplasmic role of H1.X in muscle growth and muscle function.

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

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