Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). In mammalian cells, UPR signals generated by several ER membrane resident proteins, including the bifunctional protein kinase endoribonuclease IRE1α, control cell survival and the decision to execute apoptosis. Processing of XBP1 mRNA by the RNase domain of IRE1α promotes survival of ER stress, while activation of the mitogen-activated protein kinase JNK by IRE1α late in the ER stress response promotes apoptosis. Here we show that activation of JNK in the ER stress response precedes activation of XBP1. This activation of JNK is dependent on IRE1α and TRAF2 and coincides with JNK-dependent induction of expression of several antiapoptotic genes, including cIAP1, cIAP2, XIAP, and BIRC6. ER-stressed jnk1-/-
jnk2-/- mouse embryonic fibroblasts (MEFs) display more pronounced mitochondrial permeability transition and increased caspase 3/7 activity compared to wild type MEFs. Caspase 3/7 activity is also elevated in ER-stressed ciap1-/-
ciap2-/-, and xiap-/- MEFs. These observations suggest that JNK-dependent transcriptional induction of several inhibitors of apoptosis contributes to inhibiting apoptosis early in the ER stress response.
Apoptosis; endoplasmic reticulum; IRE1; JNK; stress response; unfolded protein response
The stem-loop binding protein (SLBP) binds to the 3′ end of histone mRNA and participates in 3′-processing of the newly synthesized transcripts, which protects them from degradation, and probably also promotes their translation. In proliferating cells, translation of SLBP mRNA begins at G1/S and the protein is degraded following DNA replication. These post-transcriptional mechanisms closely couple SLBP expression to S-phase of the cell cycle, and play a key role in restricting synthesis of replication-dependent histones to S-phase. In contrast to somatic cells, replication-dependent histone mRNAs accumulate and are translated independently of DNA replication in oocytes and early embryos. We report here that SLBP expression and activity also differ in mouse oocytes and early embryos compared with somatic cells. SLBP is present in oocytes that are arrested at prophase of G2/M, where it is concentrated in the nucleus. Upon entry into M-phase of meiotic maturation, SLBP begins to accumulate rapidly, reaching a very high level in mature oocytes arrested at metaphase II. Following fertilization, SLBP remains abundant in the nucleus and the cytoplasm throughout the first cell cycle, including both G1 and G2 phases. It declines during the second and third cell cycles, reaching a relatively low level by the late 4-cell stage. SLBP can bind the histone mRNA-stem-loop at all stages of the cell cycle in oocytes and early embryos, and it is the only stem-loop binding activity detectable in these cells. We also report that SLBP becomes phosphorylated rapidly following entry into M-phase of meiotic maturation through a mechanism that is sensitive to roscovitine, an inhibitor of cyclin-dependent kinases. SLBP is rapidly dephosphorylated following fertilization or parthenogenetic activation, and becomes newly phosphorylated at M-phase of mitosis. Phosphorylation does not affect its stem-loop binding activity. These results establish that, in contrast to Xenopus, mouse oocytes and embryos contain a single SLBP. Expression of SLBP is uncoupled from S-phase in oocytes and early embryos, which indicates that the mechanisms that impose cell-cycle-regulated expression of SLBP in somatic cells do not operate in oocytes or during the first embryonic cell cycle. This distinctive pattern of SLBP expression may be required for accumulation of histone proteins required for sperm chromatin remodelling and assembly of newly synthesized embryonic DNA into chromatin.
PMID: 12415002 CAMSID: cams253
SLBP; Histone mRNA; Mouse; Oocyte; Embryo; Translational control; Cell cycle
The current model for hemoglobin ingestion and transport by intraerythrocytic Plasmodium falciparum malaria parasites shares similarities with endocytosis. However, the model is largely hypothetical, and the mechanisms responsible for the ingestion and transport of host cell hemoglobin to the lysosome-like food vacuole (FV) of the parasite are poorly understood. Because actin dynamics play key roles in vesicle formation and transport in endocytosis, we used the actin-perturbing agents jasplakinolide and cytochalasin D to investigate the role of parasite actin in hemoglobin ingestion and transport to the FV. In addition, we tested the current hemoglobin trafficking model through extensive analysis of serial thin sections of parasitized erythrocytes (PE) by electron microscopy. We find that actin dynamics play multiple, important roles in the hemoglobin transport pathway, and that hemoglobin delivery to the FV via the cytostomes might be required for parasite survival. Evidence is provided for a new model, in which hemoglobin transport to the FV occurs by a vesicle-independent process.
Plasmodium; Actin; Cytostome; Food vacuole; Hemoglobin; Malaria
Nucleolar disassembly occurs during mitosis and nucleolar stress, releasing several MDM2-interactive proteins residing in the nucleolus that share the common activity of p53 stabilization. Here, we demonstrated that mobilization of nucleostemin (NS), a cancer and stem cell-enriched nucleolar protein, plays the opposite role by stabilizing MDM2 and suppressing p53 functions. Our results showed that NS increases the protein stability and nucleoplasmic retention of MDM2, and competes with L23 for MDM2 binding. These activities are significantly elevated when NS is released into the nucleoplasm by mutations that abolish its nucleolar localization or by chemotherapeutic agents that disassemble the nucleoli. NS depletion decreases MDM2 protein, increases the transcriptional activities without changing the protein level of p53, and triggers G2/M arrest and cell death in U2OS but not in H1299 cells. This work reveals that nucleoplasmic relocation of NS during nucleolar disassembly safeguards the G2/M transit and survival of continuously dividing cells by MDM2 stabilization and p53 inhibition.
nucleostemin; MDM2; cancer; ubiquitylation; p53
Cullin-RING ligases (CRL) are ubiquitin E3s that bind substrates through variable substrate-receptor proteins. CRLs are activated by attachment of the ubiquitin-like protein NEDD8 to the Cullin subunit and DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like proteins and little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and Cullins that can only be neddylated in the presence of substrate adaptor. These DCNL-CUL-CAND1 complexes may represent “reserve” CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most Cullin subtypes, but that they are likely responsible for the neddylation of different subpopulations of any given Cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their Cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression.
ubiquitin; NEDD8; Cullin-RING Ligases; CRL
Mutations in the fibrillin-1 gene give rise to a number of heritable disorders, which are all characterized by various malformations of bone as well as manifestations in other tissues. However, the role of fibrillin-1 in the development and homeostasis of bone is not well understood. Here, we examined the role of fibrillin-1 in regulating osteoclast differentiation from primary bone-marrow-derived precursors and monocytic RAW 264.7 cells. The soluble N-terminal half of fibrillin-1 (rFBN1-N) strongly inhibited osteoclastogenesis, whereas the C-terminal half (rFBN1-C) did not. By contrast, when rFBN1-N was immobilized on calcium phosphate, it did not affect osteoclastogenesis but modulated osteoclast resorptive activity, which was evident by a larger number of smaller resorption pits. Using a panel of recombinant sub-fragments spanning rFBN1-N, we localized an osteoclast inhibitory activity to the 63 kDa subfragment rF23 comprising the N-terminal region of fibrillin-1. Osteoclastic resorption led to the generation of small fibrillin-1 fragments that were similar to those identified in human vertebral bone extracts. rF23, but not rFBN1-N, was found to inhibit the expression of cathepsin K, matrix metalloproteinase 9 and Dcstamp in differentiating osteoclasts. rFBN1-N, but not rF23, exhibited interaction with RANKL. Excess RANKL rescued the inhibition of osteoclastogenesis by rFBN1-N. By contrast, rF23 disrupted RANKL-induced Ca2+ signaling and activation of transcription factor NFATc1. These studies highlight a direct dual inhibitory role of N-terminal fibrillin-1 fragments in osteoclastogenesis, the sequestration of RANKL and the inhibition of NFATc1 signaling, demonstrating that osteoclastic degradation of fibrillin-1 provides a potent negative feedback that limits osteoclast formation and function.
PMID: 24039232 CAMSID: cams5817
Calcium signaling; Fibrillin; NFATc1; Osteoclastogenesis; RANKL
The interaction of β-actin mRNA with zipcode-binding protein 1 (ZBP1) is necessary for its localization to the lamellipod of fibroblasts and plays a crucial role in cell polarity and motility. Recently, we have shown that low ZBP1 levels correlate with tumor-cell invasion and metastasis. In order to establish a cause and effect relationship, we expressed ZBP1 in a metastatic rat mammary adenocarcinoma cell line (MTLn3) that has low endogenous ZBP1 levels and delocalized β-actin mRNA. This leads to localization of β-actin mRNA, and eventually reduces the chemotactic potential of the cells as well as their ability to move and orient towards vessels in tumors. To determine how ZBP1 leads to these two apparently contradictory aspects of cell behavior – increased cell motility but decreased chemotaxis – we examined cell motility in detail, both in cell culture and in vivo in tumors. We found that ZBP1 expression resulted in tumor cells with a stable polarized phenotype, and reduced their ability to move in response to a gradient in culture. To connect these results on cultured cells to the reduced metastatic ability of these cells, we used multiphoton imaging in vivo to examine tumor cell behavior in primary tumors. We found that ZBP1 expression actually reduced tumor cell motility and chemotaxis, presumably mediating their decreased metastatic potential by reducing their ability to respond to signals necessary for invasion.
Cellular senescence suppresses cancer by arresting cells at risk of malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation and branching morphogenesis. Furthermore, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts – the ability to alter epithelial differentiation – that might also explain the loss of tissue function and organization that is a hallmark of aging.
Epithelial to mesenchyme transition (EMT); Beta-casein; Mammary epithelial cells; Matrix metalloproteinase-3 (MMP-3); Morphogenesis; Tissue structure and function
Cullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and cullins that can only be neddylated in the presence of a substrate adaptor. These CAND–cullin–DCNL complexes might represent ‘reserve’ CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most cullin subtypes, but that they are probably responsible for the neddylation of different subpopulations of any given cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue-specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression.
Highlighted Article: The mammalian DCN-type NEDD8 E3 ligases (DCNL1–5) have overlapping and unique functions that might be related to their subcellular distribution and are mostly governed by their unique N-termini.
Ubiquitin; NEDD8; Cullin-RING ligases; CRL; SCCRO; DCUN1D
Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms μ1A and μ1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of μ1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (μ1A or μ1B) showed two distinct distribution patterns that involved Pmel17, and only μ1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking μ1B expression. Finally, we established that expression of μ1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature.
Pmel17; Plasma membrane; AP1; AP2; Melanocytes
Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 μm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid α-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.
Endosome; Membrane protein; Pinocytosis; Spectrin; Src homology domain
The C-terminus of GABA transporter 1 (GAT1, SLC6A1) is required for trafficking of the protein through the secretory pathway to reach its final destination, i.e. the rim of the synaptic specialization. We identified a motif of three hydrophobic residues (569VMI571) that was required for export of GAT1 from the ER-Golgi intermediate compartment (ERGIC). This conclusion was based on the following observations: (i) GAT1-SSS, the mutant in which 569VMI571 was replaced by serine residues, was exported from the ER in a COPII-dependent manner but accumulated in punctate structures and failed to reach the Golgi; (ii) under appropriate conditions (imposing a block at 15°C, disruption of COPI), these structures also contained ERGIC53; (iii) the punctae were part of a dynamic compartment, because it was accessible to a second anterograde cargo [the temperature-sensitive variant of vesicular stomatitis virus G protein (VSV-G)] and because GAT1-SSS could be retrieved from the punctate structures by addition of a KKxx-based retrieval motif, which supported retrograde transport to the ER. To the best of our knowledge, the VMI-motif of GAT1 provides the first example of a cargo-based motif that specifies export from the ERGIC.
GABA transporter-1; ER-to-Golgi trafficking; ERGIC
Changes in cellular behavior that cause epithelial cells to lose adhesiveness, acquire a motile, invasive phenotype and metastasize to secondary sites are complex and poorly understood. Molecules that normally function to integrate adhesive spatial information with cytoskeleton dynamics and membrane trafficking likely serve important functions in cellular transformation. One such complex is the Exocyst, which is essential for targeted delivery of membrane and secretory proteins to specific plasma membrane sites to maintain epithelial cell polarity. Upon loss of cadherin-mediated adhesion in Dunning R3327-5′A prostate tumor cells, Exocyst localization shifts from lateral membranes to tips of protrusive membrane extensions. Here, it co-localizes and co-purifies with focal complex proteins that regulate membrane trafficking and cytoskeleton dynamics. These sites are the preferred destination of post-Golgi transport vesicles ferrying biosynthetic cargo, such as α5-integrin, which mediates adhesion of cells to the substratum, a process essential to cell motility. Interference with Exocyst activity impairs integrin delivery to plasma membrane and inhibits tumor cell motility and matrix invasiveness. Localization of Exocyst, and by extension targeting of Exocyst-dependent cargo, is dependent on Ral GTPases, which control association between Sec5 and paxillin. Overexpression of Ral-uncoupled Sec5 mutants inhibited Exocyst interaction with paxillin in 5′A cells, as did RNAi-mediated reduction of either RalA or RalB. Reduction of neither GTPase significantly altered steady state levels of assembled Exocyst in these cells, but did change the observed localization of Exocyst proteins.
Like other DNA viruses, herpes simplex virus type 1 (HSV-1) interacts with components of the cellular response to DNA damage. For example, HSV-1 sequesters endogenous, uninduced, hyperphosphorylated RPA (replication protein A) away from viral replication compartments. RPA is a ssDNA-binding protein that signals genotoxic stress through the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway. The sequestration of endogenous hyperphosphorylated RPA away from replicating viral DNA suggests that HSV-1 prevents the normal ATR-signaling response. In this study we examine the spatial distribution of endogenous hyperphosphorylated RPA with respect to ATR, its recruitment factor, ATRIP, and the cellular dsDNA break marker, γH2AX, during HSV-1 infection. The accumulation of these repair factors at DNA lesions has previously been identified as an early event in signaling genotoxic stress. We show that HSV-1 infection disrupts the ATR pathway by a mechanism that prevents the recruitment of repair factors, spatially uncouples ATRIP from ATR and sequesters ATRIP and endogenous hyperphosphorylated RPA within virus-induced nuclear domains containing molecular chaperones and components of the ubiquitin proteasome. The HSV-1 immediate early protein ICP0 is sufficient to induce the redistribution of ATRIP. This is the first report that a virus can disrupt the usually tight colocalization of ATR and ATRIP.
Herpesvirus; ICP0; DNA-damage response; ATR; ATRIP; Hyperphosphorylated RPA; Phosphorylated H2AX; Chaperones; Proteasome
An important eukaryotic signal transduction pathway involves the regulation of the effector enzyme adenylate cyclase, which produces the second messenger, cAMP. Previous genetic analyses demonstrated that glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene requires the function of adenylate cyclase, encoded by the git2 gene. As mutations in git2 and in six additional git genes are suppressed by exogenous cAMP, these ‘upstream’ git genes were proposed to act to produce a glucose-induced cAMP signal. We report here that assays of cAMP levels in wild-type and various mutant S. pombe cells, before and after exposure to glucose, show that this is the case. The data suggest that the cAMP signal results from the activation of adenylate cyclase. Therefore these ‘upstream’ git genes appear to encode a glucose-induced adenylate cyclase activation pathway. Assays of cAMP on a strain carrying a mutation in the git6 gene, which acts downstream of adenylate cyclase, indicate that git6 may function to feedback regulate adenylate cyclase activity. Thus git6 may encode a cAMP-dependent protein kinase.
Schizosaccharomyces pombe; fission yeast; adenylate cyclase; cAMP; signal transduction
The Schizosaccharomyces pombe pyp1+ gene, encoding a protein tyrosine phosphatase (pyp1), was isolated as a high copy number suppressor of a mutation that results in reduced cAMP-dependent protein kinase (PKA) activity. Overexpression of pyp1+ inhibits both transcription of the fbp1 gene, which is negatively regulated by a glucose-induced activation of PKA, and sexual development, which is negatively regulated by PKA through a nitrogen- and glucose-monitoring mechanism. Overexpression of a catalytically inactive form of pyp1 has little effect on either process. Previous studies suggest that overexpression of pyp1+ results in a mitotic delay by positively regulating wee1 activity. We show that pyp1 repression of fbp1 transcription is independent of wee1. The direct role of the pyp1 protein is to dephosphorylate and inactivate the sty1/spc1 mitogen-activated protein kinase (MAPK) that is activated by the wis1 MAPK kinase. As overexpression of pyp1+ has no further effect upon the mitotic delay observed in a wis1 deletion strain, the role of pyp1 appears to be restricted to negative regulation of the sty1/spc1 MAPK. This study indicates that pyp1 negatively regulates fbp1 transcription, sexual development and mitosis by inactivation of the sty1/spc1 MAPK, but that bifurcations downstream of the MAPK separate these processes as seen by the differential role for the wee1 gene.
Schizosaccharomyces pombe; Fission yeast; Nutrient monitoring; fbp1; pyp1; wis1; Tyrosine phosphatase; Meiosis; PKA
ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval via interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here we show that also conserved histidines in the C-terminal tail regulate ERp44 in vivo. Mutants lacking these histidines are hyperactive in retaining substrates. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon over-expression of different partners. The ensuing gradients may help optimising folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes.
Throughout normal development, and in aberrant conditions such as cancer, cells divide by a process called cytokinesis. Most textbooks suggest that animal cells execute cytokinesis using an actomyosin-containing contractile ring, whereas plant cells generate a new cell wall by the assembly of a novel membrane compartment using vesicle-trafficking machinery in an apparently distinct manner. Recent studies have shown that cytokinesis in animal and plant cells may not be as distinct as these models imply – both have an absolute requirement for vesicle traffic. Moreover, some of the key molecular components of cytokinesis have been identified, many of which are proteins that function to control membrane traffic. Here, we review recent advances in this area.
Cytokinesis; ER; Endosome; Secretory pathway; Membrane traffic
Sorting and recycling of endocytosed proteins are required for proper cellular function and growth. Internalized receptors either follow a fast constitutive recycling pathway, returning to the cell surface directly from the early endosomes, or a slow pathway that involves transport via perinuclear recycling endosomes. Slow recycling pathways are thought to play a key role in directing recycling proteins to specific locations on cell surfaces, such as the leading edges of motile cells. These pathways are regulated by various Rab GTPases, such as Rab4 and Rab11. Here we characterize the role of Rip11/FIP5, a known Rab11-binding protein, in regulating endocytic recycling. We use a combination of electron and fluorescent microscopy with siRNA-based protein knockdown to show that Rip11/FIP5 is present at the peripheral endosomes, where it regulates the sorting of internalized receptors to a slow recycling pathway. We also identify kinesin II as a Rip11/FIP5-binding protein and show that it is required for directing endocytosed proteins into the same recycling pathway. Thus, we propose that the Rip11/FIP5-kinesin-II complex has a key role in the routing of internalized receptors through the perinuclear recycling endosomes.
Endosomes; Rab11 GTPase; Kinesin II
The Lamina-associated polypeptide, Emerin, MAN1 - (LEM) domain defines a group of nuclear proteins, which bind chromatin through interaction of the LEM motif with the conserved DNA cross-linking protein, Barrier-to-Auto-Integration factor (BAF). Here, we describe a novel LEM protein, annotated in databases as “Ankyrin and LEM domain containing protein 1” (ANKLE1). We show that Ankle1 is conserved in metazoans and contains a unique C-terminal GIY-YIG motif that confers endonuclease activity in vitro and in vivo. In mammals, Ankle1 is predominantly expressed in hematopoietic tissues. While most characterized LEM proteins are components of the inner nuclear membrane, ectopic Ankle1 shuttles between cytoplasm and nucleus, and Ankle1 enriched in the nucleoplasm induces DNA cleavage and DNA damage response. This activity requires both the catalytic C-terminal GIY-YIG domain and the LEM motif, which binds chromatin via BAF. Hence, Ankle1 represents a novel LEM-protein with a GIY-YIG type endonuclease activity in higher eukaryotes.
chromatin; DNA damage; GIY-YIG endonuclease; LEM-domain; nuclear envelope
A-type lamins are components of the nuclear lamina, a filamentous network of the nuclear envelope in metazoans that supports nuclear architecture. In addition, lamin A/C can also be found in the nuclear interior. This nucleoplasmic lamin pool is soluble in physiological buffer, depends on the presence of the lamin-binding protein, Lamina-associated polypeptide 2α (LAP2α) and regulates cell cycle progression in tissue progenitor cells. ΔK32 mutations in A-type lamins cause severe congenital muscle disease in humans and a muscle maturation defect in LmnaΔK32/ΔK32 knock-in mice. At molecular level, mutant ΔK32 lamin A/C protein levels were reduced and all mutant lamin A/C was soluble and mislocalized to the nucleoplasm. To test the role of LAP2α in nucleoplasmic ΔK32 lamin A/C regulation and functions, we deleted LAP2α in LmnaΔK32/ΔK32 knock-in mice. In double mutant mice the LmnaΔK32/ΔK32- linked muscle defect was unaffected. LAP2α interacted with mutant lamin A/C, but unlike wild-type lamin A/C, the intranuclear localization of ΔK32 lamin A/C was not affected by loss of LAP2α. In contrast, loss of LAP2α in LmnaΔK32/ΔK32 mice impaired the regulation of tissue progenitor cells like in lamin A/C wild type animals. These data indicate that a LAP2α-independent assembly defect of ΔK32 lamin A/C is predominant for the mouse pathology, while the LAP2α-linked functions of nucleoplasmic lamin A/C in the regulation of tissue progenitor cells are not affected in LmnaΔK32/ΔK32 mice.
congenital muscular dystrophy; nuclear envelope; lamin A/C; lamina associated polypeptide 2α; nucleoplasmic lamins
The nuclear envelope consists of inner and outer nuclear membranes. While the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane represents a unique membranous environment containing specific proteins. The mechanisms of integral inner nuclear membrane protein degradation are unknown. Here we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome and independent of the vacuole exhibiting a half-life of ≈ 45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistently, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.
ERAD; nuclear membrane; nuclear proteasome; protein degradation; ubiquitylation
Chromatin-related functions are associated with spatial organization in the nucleus. We have investigated the relationship between the enhancer-blocking activity and subnuclear localization of the Drosophila melanogaster suHw insulator. Using fluorescent in situ hybridization, we observed that genomic loci containing the gypsy retrotransposon were distributed closer to the nuclear periphery than regions without the gypsy retrotransposon. However, transgenes containing a functional 340 bp suHw insulator did not exhibit such biased distribution towards the nuclear periphery, which suggests that the suHw insulator sequence is not responsible for the peripheral localization of the gypsy retrotransposon. Antibody stains showed that the two proteins essential for the suHw insulator activity, SUHW and MOD(MDG4), are not restricted to the nuclear periphery. The enhancer-blocking activity of suHw remained intact under the heat shock conditions, which was shown to disrupt the association of gypsy, SUHW and MOD(MDG4) with the nuclear periphery. Our results indicate that the suHw insulator can function in the nuclear interior, possibly through local interactions with chromatin components or other nuclear structures.
Chromatin boundary; Insulator; suHw; SuHw; Gypsy; Drosophila