Dpy19l1, a multi-transmembrane protein, regulates the radial migration of glutamatergic neurons in the developing cerebral cortex

During corticogenesis, the regulation of neuronal migration is crucial for the functional organization of the neocortex. Glutamatergic neurons are major excitatory components of the mammalian neocortex. In order to elucidate the specific molecular mechanisms underlying their development, we used single-cell microarray analysis to screen for mouse genes that are highly expressed in developing glutamatergic neurons. We identified dpy-19-like 1 (Dpy19l1), a homolog of C. elegans dpy-19, which encodes a putative multi-transmembrane protein shown to regulate directed migration of Q neuroblasts in C. elegans. At embryonic stages Dpy19l1 is highly expressed in glutamatergic neurons in the mouse cerebral cortex, whereas in the subpallium, where GABAergic neurons are generated, expression was below detectable levels. Downregulation of Dpy19l1 mediated by shRNA resulted in defective radial migration of glutamatergic neurons in vivo, which was restored by the expression of shRNA-insensitive Dpy19l1. Many Dpy19l1-knockdown cells were aberrantly arrested in the intermediate zone and the deep layer and, additionally, some extended single long processes towards the pial surface. Furthermore, we observed defective radial migration of bipolar cells in Dpy19l1-knockdown brains. Despite these migration defects, these cells correctly expressed Cux1, which is a marker for upper layer neurons, suggesting that Dpy19l1 knockdown results in migration defects but does not affect cell type specification. These results indicate that Dpy19l1 is required for the proper radial migration of glutamatergic neurons, and suggest an evolutionarily conserved role for the Dpy19 family in neuronal migration.     

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

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.     

A three-residue signal confers localization of a reporter protein in the inner nuclear membrane

By alignment to the carboxy-terminal-deduced aa sequence of human cytomegalovirus glycoprotein B (gB), conserved hexameric aa motifs with putative function for localization in the inner nuclear membrane (INM) were identified in the nucleoplasmic tails of herpes simplex virus type 1 gB and of the cellular lamin B receptor. Fusion of the respective hexamers to CD8 as a reporter redirected transport of the chimeras into the INM, suggesting their functioning as modular signal motifs. Consecutive experiments showed that the three-residue motif RxR represents a consensus sequence which is sufficient for localization of the CD8 reporter in the INM.     

Inactivation of YME2/RNA12, which encodes an integral inner mitochondrial membrane protein, causes increased escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae.

Inactivation of the yeast nuclear gene YMe2 causes an increased rate of DNA escape from mitochondria to the nucleus. Mutations in yme2 also show genetic interactions with yme1, a second gene that affects DNA escape from mitochondria to the nucleus. The yme1 cold-sensitive growth phenotype is suppressed by yme2 mutations. In addition, yme1 yme2 double mutants exhibit a synthetic growth defect on ethanol-glycerol medium at 30 degrees C. YME2 was isolated by complementation of the synthetic growth defect of yme1 yme2 strains and was found to be identical with the previously cloned RNA12 gene. The dominant temperature-sensitive mutation RNA12-1 prevents growth of yeast cells at 37 degrees C. YME2 encodes a protein with a predicted molecular weight of 96,681 and is an integral inner mitochondrial membrane protein. The larger carboxyl-terminal domain of the YME2 gene product faces the intermembrane space. Null alleles of yme2 display the same genetic interactions with yme1 and high rate of DNA escape from mitochondria as do the originally isolated yme2 mutant strains. Disruption of yme2 causes a strain-dependent growth defect on nonfermentable carbon sources.     

Targeting of the SUN protein Mps3 to the inner nuclear membrane by the histone variant H2A.Z

Understanding the relationship between chromatin and proteins at the nuclear periphery, such as the conserved SUN family of inner nuclear membrane (INM) proteins, is necessary to elucidate how three-dimensional nuclear architecture is established and maintained. We found that the budding yeast SUN protein Mps3 directly binds to the histone variant H2A.Z but not other histones. Biochemical and genetic data indicate that the interaction between Mps3 and H2A.Z requires the Mps3 N-terminal acidic domain and unique sequences in the H2A.Z N terminus and histone-fold domain. Analysis of binding-defective mutants showed that the Mps3-H2A.Z interaction is not essential for any previously described role for either protein in nuclear organization, and multiple lines of evidence suggest that Mps3-H2A.Z binding occurs independently of H2A.Z incorporation into chromatin. We demonstrate that H2A.Z is required to target a soluble Mps3 fragment to the nucleus and to localize full-length Mps3 in the INM, indicating that H2A.Z has a novel chromatin-independent function in INM targeting of SUN proteins.     

Monoclonal antibodies to NTF2 inhibit nuclear protein import by preventing nuclear translocation of the GTPase Ran

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.     

Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane

Newly synthesized membrane proteins are constantly sorted from the endoplasmic reticulum (ER) to various membranous compartments. How proteins specifically enrich at the inner nuclear membrane (INM) is not well understood. We have established a visual in vitro assay to measure kinetics and investigate requirements of protein targeting to the INM. Using human LBR, SUN2, and LAP2β as model substrates, we show that INM targeting is energy-dependent but distinct from import of soluble cargo. Accumulation of proteins at the INM relies on both a highly interconnected ER network, which is affected by energy depletion, and an efficient immobilization step at the INM. Nucleoporin depletions suggest that translocation through nuclear pore complexes (NPCs) is rate-limiting and restricted by the central NPC scaffold. Our experimental data combined with mathematical modeling support a diffusion-retention–based mechanism of INM targeting. We experimentally confirmed the sufficiency of diffusion and retention using an artificial reporter lacking natural sorting signals that recapitulates the energy dependence of the process in vivo.     

The translocon Sec61beta localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus

Accumulating evidence indicates that endocytosis plays an essential role in the nuclear transport of the ErbB family members, such as epidermal growth factor receptor (EGFR) and ErbB-2. Nevertheless, how full-length receptors embedded in the endosomal membrane pass through the nuclear pore complexes and function as non-membrane-bound receptors in the nucleus remains unclear. Here we show that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the inner nuclear membrane (INM) through the nuclear pore complexes, remaining in a membrane-bound environment. We further find that importin β regulates EGFR nuclear transport to the INM in addition to the nucleus/nucleoplasm. Unexpectedly, the well known endoplasmic reticulum associated translocon Sec61β is found to reside in the INM and associate with EGFR. Knocking down Sec61β expression reduces EGFR level in the nucleoplasm portion and accumulates it in the INM portion. Thus, the Sec61β translocon plays an unrecognized role in the release of the membrane-anchored EGFR from the lipid bilayer of the INM to the nucleus. The newly identified Sec61β function provides an alternative pathway for nuclear transport that can be utilized by membrane-embedded proteins such as full-length EGFR.     

Activation of endoplasmic reticulum stress in premature aging via the inner nuclear membrane protein SUN2

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Afaf, a novel vesicle membrane protein, is related to acrosome formation in murine testis

As a cell-specific organelle, acrosome (Acr) and its formation are an important event for spermiogenesis. However, the Acr formation is far more complicated than has been proposed. In this study, we have cloned a novel membrane protein Afaf (Acr formation associated factor) that was expressed abundantly in the round spermatids, localized in the inner and outer membrane of forming Acrs, and declined in the maturing Acrs. In the transfected Hela cells, Afaf protein was localized in the plasma membrane, EEA1-positive early endosomes (EEs) and occasionally in the nuclei. Therefore, we propose that EEs and plasma membrane may be also directly involved in the Acr biogenesis.     

Recruitment of protein phosphatase 1 to the nuclear envelope by A-kinase anchoring protein AKAP149 is a prerequisite for nuclear lamina assembly

Subcellular targeting of cAMP-dependent protein kinase (protein kinase A [PKA]) and of type 1 protein phosphatase (PP1) is believed to enhance the specificity of these enzymes. We report that in addition to anchoring PKA, A-kinase anchoring protein AKAP149 recruits PP1 at the nuclear envelope (NE) upon somatic nuclear reformation in vitro, and that PP1 targeting to the NE is a prerequisite for assembly of B-type lamins. AKAP149 is an integral membrane protein of the endoplasmic reticulum/NE network. The PP1-binding domain of AKAP149 was identified as K(153)GVLF(157). PP1 binds immobilized AKAP149 in vitro and coprecipitates with AKAP149 from purified NE extracts. Affinity isolation of PP1 from solubilized NEs copurifies AKAP149. Upon reassembly of somatic nuclei in interphase extract, PP1 is targeted to the NE. Targeting is inhibited by a peptide containing the PP1-binding domain of AKAP149, abolished in nuclei assembled with membranes immunodepleted of AKAP149, and restored after reincorporation of AKAP149 into nuclear membranes. B-type lamins do not assemble into a lamina when NE targeting of PP1 is abolished, and is rescued upon recruitment of PP1 to the NE. We propose that kinase and phosphatase anchoring at the NE by AKAP149 plays in a role in modulating nuclear reassembly at the end of mitosis.     

Analysis of the localization and topology of nurim, a polytopic protein tightly associated with the inner nuclear membrane

Nurim is an inner nuclear membrane (INM) protein that was first isolated in a visual screen for nuclear envelope-localizing proteins. Nurim lacks an N-terminal domain characteristic of other INM proteins examined to date and may represent a class of proteins that localize to the INM by a distinct mechanism. To further characterize this protein, we constructed nurim-green fluorescent protein fusions and analyzed aspects of localization, biochemistry, and membrane topology. Results from immunoprobing and protease protection assays together with other analyses indicate that nurim (total length of 262 residues) is a six transmembrane-spanning protein and contains a hairpin turn in its C-terminal transmembrane domain, resulting in the N and C termini residing on the same side of the membrane. A loop region between the fourth and fifth transmembrane domains is exposed toward the nucleoplasm and contains a region accessible for site-specific endoproteinase cleavage. In biochemical fractionation, nurim remained extremely tightly bound to nuclear fractions and was released in significant quantities only in the presence of 4 m urea. Under conditions in which nuclear lamins were completely extracted, a significant population of nurim remained resistant to solubilization. This tight binding requires the C-terminal region of the protein. DNase treatment only marginally influenced its retention characteristics in nuclei. Results from consideration of sequence alignments and identification of specific topological features of nurim indicate that it may possess enzymic function. These results are discussed with reference to the retention mechanism and possible nuclear function of nurim.     

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.     

MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin

The "MAN antigens" are polypeptides recognized by autoantibodies from a patient with a collagen vascular disease and localized to the nuclear envelope. We now show that one of the human MAN antigens termed MAN1 is a 82.3-kDa protein with an amino-terminal domain followed by two hydrophobic segments and a carboxyl-terminal tail. The MAN1 gene contains seven protein-coding exons and is assigned to human chromosome 12q14. Its mRNA is approximately 5.5 kilobases and is detected in several different cell types that were examined. Cell extraction experiments show that MAN1 is an integral membrane protein. When expressed in transfected cells, MAN1 is exclusively targeted to the nuclear envelope, consistent with an inner nuclear membrane localization. Protein sequence analysis reveals that MAN1 shares a conserved globular domain of approximately 40 amino acids, which we term the LEM module, with inner nuclear membrane proteins lamina-associated polypeptide 2 and emerin. The LEM module is also present in two proteins of Caenorhabditis elegans. These results show that MAN1 is an integral protein of the inner nuclear membrane that shares the LEM module with other proteins of this subcellular localization.     

Herpes simplex virus infection induces phosphorylation and delocalization of emerin, a key inner nuclear membrane protein

The inner nuclear membrane (INM) contains specialized membrane proteins that selectively interact with nuclear components including the lamina, chromatin, and DNA. Alterations in the organization of and interactions with INM and lamina components are likely to play important roles in herpesvirus replication and, in particular, exit from the nucleus. Emerin, a member of the LEM domain class of INM proteins, binds a number of nuclear components including lamins, the DNA-bridging protein BAF, and F-actin and is thought to be involved in maintaining nuclear integrity. Here we report that emerin is quantitatively modified during herpes simplex virus (HSV) infection. Modification begins early in infection, involves multiple steps, and is reversed by phosphatase treatment. Emerin phosphorylation during infection involves one or more cellular kinases but can also be influenced by the US3 viral kinase, a protein whose function is known to be involved in HSV nuclear egress. The results from biochemical extraction analyses and from immunofluorescence of the detergent-resistant population demonstrate that emerin association with the INM significantly reduced during infection. We propose that the induction of emerin phosphorylation in infected cells may be involved in nuclear egress and uncoupling interactions with targets such as the lamina, chromatin, or cytoskeletal components.     

Removal of a hydrophobic domain within the mature portion of a mitochondrial inner membrane protein causes its mislocalization to the matrix.

We have examined the import and intramitochondrial localization of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. The results of studies on the import of subunit Va derivatives carrying altered presequences suggest that the uptake of this protein is highly efficient. We found that a presequence of only 5 amino acids (Met-Leu-Ser-Leu-Arg) could direct the import and localization of subunit Va with wild-type efficiency, as judged by several different assays. We also found that subunit Va could be effectively targeted to the mitochondrial inner membrane with a heterologous presequence that failed to direct import of its cognate protein. The results presented here confirmed those of an earlier study and showed clearly that the information required to "sort" subunit Va to the inner membrane resides in the mature protein sequence, not within the presequence per se. We present additional evidence that the aforementioned sorting information is contained, at least in part, in a hydrophobic stretch of 22 amino acids residing within the C-terminal third of the protein. Removal of this domain caused subunit Va to be mislocalized to the mitochondrial matrix.     

The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins.

p58, also referred to as the lamin B receptor, is an intrinsic protein of the inner nuclear membrane that binds in vitro to lamin B. Previous studies have demonstrated that p58 is phosphorylated in vivo and removal of its phosphate moieties affects lamin B binding. Using affinity-purified antipeptide antibodies, we have now immunoisolated p58 from bird erythrocyte lysates under isotonic, non-denaturing conditions. Analysis of the immunopurified material shows that five distinct proteins are tightly and specifically associated with p58. Two of these polypeptides can be identified as nuclear lamins A and B. The immunoisolate also contains a kinase activity that phosphorylates p58 in vivo and in vitro, exclusively at serine residues, as indicated by phosphoamino acid analysis and two-dimensional phosphopeptide mapping. Cell fractionation experiments and in vitro phosphorylation assays demonstrate that the p58 kinase resides in the nuclear envelope and is distinct from protein kinase A and cdc2 kinase, for both of which p58 is an in vitro substrate. These data suggest that p58 is interacting in vivo with a p58 kinase and the nuclear lamins.