Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2.

Polymerase basic protein 2 (PB2), a component of the influenza virus polymerase complex, when expressed alone from cloned cDNA in the absence of other influenza virus proteins, is transported into the nucleus. In this study, we have examined the nuclear translocation signal of PB2 by making deletions and mutations in the PB2 sequence. Our studies showed that two distant regions in the polypeptide sequence were involved in the nuclear translocation of PB2. In one region, four basic residues (K-736 R K R) played a critical role in the nuclear translocation of PB2, since the deletion or mutation of these residues rendered the protein totally cytoplasmic. However, seven residues (M K R K R N S) of this region, including the four basic residues, failed to translocate a cytoplasmic reporter protein into the nucleus, suggesting that these sequences were necessary but not sufficient for nuclear translocation. Deletion of another region (amino acids 449 to 495) resulted in a mutant protein which was cytoplasmic with a perinuclear distribution. This novel phenotype suggests that a perinuclear binding step was involved prior to translocation of PB2 across the nuclear pore and that a signal might be involved in perinuclear binding. Possible involvement of these two signal sequences in the nuclear localization of PB2 is discussed.     

Identification of domains involved in nuclear uptake and histone binding of protein N1 of Xenopus laevis.

The karyophilic protein N1 (590 amino acids) is an abundant soluble protein of the nuclei of Xenopus laevis oocytes where it forms defined complexes with histones H3 and H4. The amino acid sequence of this protein, as deduced from the cDNA, reveals a putative nuclear targeting signal as well as two acidic domains which are candidates for the interaction with histones. Using two different histone binding assays in vitro we have found that the deletion of the larger acidic domain reduces histone binding drastically to a residual value of approximately 15% of the complete molecule, whereas removal of the smaller acidic domain only slightly reduces histone complex formation in solution, but infers more effectively with binding to immobilized histones. In the primary structure of the protein both histone-binding domains are distant from the conspicuous nuclear accumulation signal sequence (residues 531-537) close to the carboxy terminus which is very similar to the SV40 large T-antigen nuclear targeting sequence. Using a series of N1 mutants altered by deletions or point mutations we show that this signal is required but not sufficient for nuclear accumulation of protein N1. The presence of an additional, more distantly related signal sequence in position 544-554 is also needed to achieve a level of nuclear uptake equivalent to that of the wild-type protein. Results obtained with point mutations support the concept of two nuclear targeting sequences and emphasize the importance of specific lysine and arginine residues in these signal sequences.     

Carbohydrate binding protein 35. Complementary DNA sequence reveals homology with proteins of the heterogeneous nuclear RNP

The complete nucleotide sequence of a cDNA clone for carbohydrate binding protein 35, a galactose-specific lectin identified in the nucleus of mouse 3T3 fibroblasts, has been determined. The deduced amino acid sequence suggests that the protein consists of two domains: (a) an amino-terminal portion that is homologous to certain regions of proteins of the heterogeneous nuclear ribonucleoprotein complex, and (b) a carboxyl-terminal portion that is homologous to beta-D-galactoside-specific lectins isolated from a number of animal tissues. This two-domain motif is reminiscent of several DNA- and RNA-binding proteins.     

Function of two discrete regions is required for nuclear localization of polymerase basic protein 1 of A/WSN/33 influenza virus (H1 N1).

Polymerase basic protein 1 (PB1) of influenza virus (A/WSN/33), when expressed from cloned cDNA in the absence of other viral proteins, accumulates in the nucleus. We have examined the location and nature of the nuclear localization signal of PB1 by using deletion mutants and chimeric constructions with chicken muscle pyruvate kinase, a cytoplasmic protein. Our studies showed some novel features of the nuclear localization signal of PB1. The signal was present internally within residues 180 to 252 of PB1. Moreover, unlike most nuclear localization signals, it was not a single stretch of contiguous amino acids. Instead, it possessed two discontinuous regions separated by an intervening sequence which could be deleted without affecting its nuclear localization property. On the other hand, deletion of either of the two signal regions rendered the protein cytoplasmic, indicating that the function of both regions is required for nuclear localization and that one region alone is not sufficient. Both of these signal regions contained short stretches of basic residues. Possible ways by which this novel bipartite signal can function in nuclear localization are discussed.     

The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis.

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking.     

The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking. Copyright 2001 Academic Press.     

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.     

NuMA: an unusually long coiled-coil related protein in the mammalian nucleus

A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.     

A novel alpha-helical protein,specific to and highly conserved in plants,is associated with the nuclear matrix fraction

A cDNA for a novel plant protein was isolated from tomato. Nuclear Matrix Protein 1 (NMP1) is a ubiquitously expressed 36 kDa protein, which has no homologues in animals and fungi, but is highly conserved among flowering and non-flowering plants, including gymnosperms, moss, and the liverwort Marchantia polymorpha. NMP1 is predominantly alpha-helical with multiple stretches of short amphipathic regions. Cell fractionation, immunofluorescence, and GFP localization experiments showed that NMP1 is located both in the cytoplasm and nucleus and that the nuclear fraction is associated with the nuclear matrix. NMP1 is a candidate for a plant-specific structural protein with a function both in the nucleus and cytoplasm.