Nuclear Envelope Fraction Research Articles

Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells

ABSTRACTThe double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) – the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) – a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

Isolation, Proteomic Analysis, and Microscopy Confirmation of the Liver Nuclear Envelope Proteome.

Nuclei can be relatively easily extracted from homogenized liver due to the softness of the tissue and crudely separated from other cellular organelles by low-speed centrifugation due to the comparatively large size of nuclei. However, further purification is complicated by nuclear envelope continuity with the endoplasmic reticulum, invaginations containing mitochondria, and connections to the cytoskeleton. Subsequent purification to nuclear envelopes is additionally confounded by connections of inner nuclear membrane proteins to chromatin. For these reasons, it is necessary to confirm proteomic identification of nuclear envelope proteins by testing targeting of individual proteins. The proteomic identification of nuclear envelope fractions is affected by the tendencies of transmembrane proteins to have extreme isoelectric points, strongly hydrophobic peptides, posttranslational modifications, and a propensity to aggregate, thus making proteolysis inefficient. To circumvent these problems, we have developed a MudPIT approach that uses multiple extractions and sequential proteolysis to increase identifications. Here we describe methods for isolating nuclear envelopes, determining their proteome by MudPIT, and confirming their targeting to the nuclear periphery by microscopy.

The discovery of a Werner helicase interacting protein (WHIP) association with the nuclear pore complex

The gateway for molecular trafficking between the cytoplasm and the nucleus is the Nuclear Pore Complex (NPC). Through mass spectral analysis of the isolated Nuclear Pore Nup107-160 subcomplex, we discovered an in vivo interaction with Werner's Helicase Interacting Protein 1, (WRNIP1 or WHIP). WHIP was originally identified as a binding partner of Werner protein (WRN), which functions to maintain genome stability and is responsible for the progeria disease, Werner syndrome. We established the reciprocal isolation of Nup107 by α-WHIP. WHIP was found in purified Nuclear Envelope (NE) fractions treated with DNase/RNase/Heparin. We demonstrated by immunofluorescence microscopy that WHIP is located at the nuclear rim as well as punctate regions in the nuclear matrix. Ultimately, synchronized cells show a dynamic association between WHIP and the Nup107-160 subcomplex through the cell cycle without an interaction with WRN. We thus identify WHIP as a partner/component of the NE/NPC and set forth to investigate a role for the protein positioned at the NPC.

Comparative proteomic analyses of the nuclear envelope and pore complex suggests a wide range of heretofore unexpected functions

Since the discovery of several inherited diseases linked to the nuclear envelope the number of functions ascribed to this subcellular organelle has skyrocketed. However the molecular pathways underlying these functions are not clear in most cases, perhaps because of missing components. Several recent proteomic analyses of the nuclear envelope and nuclear pore complex proteomes have yielded not only enough missing components to potentially elucidate these pathways, but suggest an exponentially greater number of functions at the nuclear periphery than ever imagined. Many of these functions appear to derive from recapitulation of pathways utilized at the plasma membrane and from other membrane systems. Additionally, many proteins identified in the comparative nuclear envelope studies have sequence characteristics suggesting that they might also contribute to nuclear pore complex functions. In particular, the striking enrichment for proteins in the nuclear envelope fractions that carry phenylalanine-glycine (FG) repeats may be significant for the mechanism of nuclear transport. In retrospect, these findings are only surprising in context of the notion held for many years that the nuclear envelope was only a barrier protecting the genome. In fact, it is arguably the most complex membrane organelle in the cell.

Nuclear and nuclear envelope localization of dystrophin Dp71 and dystrophin‐associated proteins (DAPs) in the C2C12 muscle cells: DAPs nuclear localization is modulated during myogenesis

Dystrophin and dystrophin-associated proteins (DAPs) form a complex around the sarcolemma, which gives stability to the sarcolemma and leads signal transduction. Recently, the nuclear presence of dystrophin Dp71 and DAPs has been revealed in different non-muscle cell types, opening the possibility that these proteins could also be present in the nucleus of muscle cells. In this study, we analyzed by Immunofluorescence assays and Immunoblotting analysis of cell fractions the subcellular localization of Dp71 and DAPs in the C(2)C(12) muscle cell line. We demonstrated the presence of Dp71, alpha-sarcoglycan, alpha-dystrobrevin, beta-dystroglycan and alpha-syntrophin not only in plasma membrane but also in the nucleus of muscle cells. In addition, we found by Immunoprecipitation assays that these proteins form a nuclear complex. Interestingly, myogenesis modulates the presence and/or relative abundance of DAPs in the plasma membrane and nucleus as well as the composition of the nuclear complex. Finally, we demonstrated the presence of Dp71, alpha-sarcoglycan, beta-dystroglycan, alpha-dystrobrevin and alpha-syntrophin in the C(2)C(12) nuclear envelope fraction. Interestingly, alpha-sarcoglycan and beta-dystroglycan proteins showed enrichment in the nuclear envelope, compared with the nuclear fraction, suggesting that they could function as inner nuclear membrane proteins underlying the secondary association of Dp71 and the remaining DAPs to the nuclear envelope. Nuclear envelope localization of Dp71 and DAPs might be involved in the nuclear envelope-associated functions, such as nuclear structure and modulation of nuclear processes.

Reverse Recruitment of DNA

Menon et al. have proposed an intriguing model for transcriptional activation in yeast in which DNA moves to a platform of assembled components of the transcriptional machinery anchored to nuclear pore proteins. In Saccharomyces cerevisiae , Rap1 (repressor/activator protein 1), Gcr1, and Gcr2 form a complex that activates transcription of glycolytic and translational component genes. Synthetic genetic array analysis, in which the combined effects on synthetic growth of deletion of the gene encoding Gcr1 or Gcr2 and deletion of each of about 4700 different yeast genes were assessed, revealed a functional link between the Rap1 complex and components of the nucleoporin Nup84 nuclear pore subcomplex. Western analysis indicated that Rap1, Gcr1, and Gcr2 were enriched in nuclear envelope fractions, whereas coimmunoprecipitation demonstrated association of Gcr1 and Gcr2 with Nup84 and two other nuclear pore proteins and with a β-importin associated with the nuclear pore complex. Fusion proteins in which Nup84, other members of the Nup84 subcomplex, or two other nucleoporins were combined with a heterologous DNA binding domain transcriptionally activated a gene reporter. Fluorescent labeling of Nup84 and Nup85 fusion proteins indicated that this transcriptional activation occurred at the nuclear periphery. Transcriptional activation by nucleoporins depended on expression of gcr1 and gcr2 , whereas transcriptional activation by Gcr1 and Gcr2 was impaired by the lack of nup84 . Thus, the authors conclude that transcriptional activation by Rap1 occurs through a protein complex at the nuclear periphery and speculate on the possibility that transcriptional activation by nucleoporins—and "reverse recruitment" of DNA--may be widespread phenomena. B. B. Menon, N. J. Sarma, S. Pasula, S. J. Deminoff, K. A. Willis, K. E. Barbara, B. Andrews, G. M. Santangelo, Reverse recruitment: The Nup84 nuclear pore subcomplex mediates Rap1/Gcr1/Gcr2 transcriptional activation. Proc. Natl. Acad. Sci. U.S.A. 102 , 5749-5754 (2005). [Abstract] [Full Text]

Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography–thermospray mass spectrometry

I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC–thermospray mass spectrometry. The HPLC–thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa + and MH +–H 2O ions. As the other minor ions, MK +, MH + and m/ z 282 ions were detected. Although the preponderance of MNa + ions compared with the MH +–H 2O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH +–H 2O ions based on the elimination of the hydroxyl group introduced at the α-position of acyl-portion compared with the MNa + ions was detected in α-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor ( f) of each ceramide was calculated as follows; N-oleoyl- d-sphingosine ( f=0.45), N-palmitoyl- d-sphingosine ( f=0.40), N-stearoyl- d-sphingosine ( f=0.39), N-nervonoyl- d-sphingosine ( f=0.39) and N-lignoceroyl- d-sphingosine ( f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100 000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa +/MH +–H 2O ions corresponding to several ω-hydroxyacyl-ceramides were detected.

Phosphatidic acid is the prominent product of endogenous neuronal nuclear lipid phosphorylation, an activity enhanced by sphingosine, linked to phospholipase C and associated with the nuclear envelope

Using endogenous lipid substrates, assays of lipid phosphorylation indicated that neuronal nuclei had a considerable superiority in phosphatidic acid (PA) formation when compared with homogenates and other subfractions of cerebral cortex. This predominance of neuronal nuclear PA labelling was linked to a sizable pool of nuclear diacylglycerols that expanded significantly with incubation. PA was also the dominant product of neuronal nuclear lipid phosphorylation reactions. Nuclear envelope preparations and the parent neuronal nuclei showed specific rates of PA formation that were comparable, based upon membrane phospholipid contents. As well, using an exogenous diacylglycerol substrate, the distribution of diacylglycerol kinase activities closely followed phospholipid contents of subfractions derived from the neuronal nucleus during envelope preparation. This evidence suggested an association between diacylglycerol kinase and the neuronal nuclear envelope. Nuclear PA formation increased in the presence of sphingosine, while sphingosine decreased PA formation in other subfractions. Likely sphingosine exerted its effect on nuclear diacylglycerol kinase, as sphingosine did not elevate levels of nuclear diacylglycerols. Phosphoinositidase C was present in the nuclei and inhibitors of this enzyme did decrease PA formation, indicating diacylglycerols from inositides as substrates for nuclear diacylglycerol kinase. The nuclear envelope fraction had a considerably lower specific phosphoinositidase C activity than the parent nuclei, and showed an activation of PA formation by sphingosine, but a less efficient handling of the exogenous diacylglycerol substrate. It is possible that phosphoinositidase C and diacylglycerol kinase are closely situated within the neuronal nuclei, and a loss of the former activity may compromise the latter.

Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear membrane.

The nuclear envelope (NE) is one of the least characterized structures of eukaryotic cells. The study of its functional roles is hampered by the small number of proteins known to be specifically located to it. Here, we present a comprehensive characterization of the NE proteome. We applied different fractionation procedures and isolated protein subsets derived from distinct NE compartments. We identified 148 different proteins by 16-benzyl dimethyl hexadecyl ammonium chloride (16-BAC) gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry; among them were 19 previously unknown or noncharacterized. The identification of known proteins in particular NE fractions enabled us to assign novel proteins to NE substructures. Thus, our subcellular proteomics approach retains the screening character of classical proteomic studies, but also allows a number of predictions about subcellular localization and interactions of previously noncharacterized proteins. We demonstrate this result by showing that two novel transmembrane proteins, a 100-kDa protein with similarity to Caenorhabditis elegans Unc-84A and an unrelated 45-kDa protein we named LUMA, reside in the inner nuclear membrane and likely interact with the nuclear lamina. The utility of our approach is not restricted to the investigation of the NE. Our approach should be applicable to the analysis of other complex membrane structures of the cell as well.

Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase.

Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of Mad1 (HsMAD1), we have begun to characterize this protein in mammalian cells. HsMad1 is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find HsMad1 associated with HsMad2. Association with p55CDC, a protein previously shown to bind HsMad2, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, HsMad1 and HsMAD2 were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. HsMad1 was identified serendipitously by its binding to a viral protein, HTLV-1 Tax, which affects transcription of viral and human proteins. The localization of HsMad1 to nuclear pore complexes suggests an alternate, non-mitotic role for the Mad1/Tax interaction in the viral transformation of cells.

Omega-hydroxylation activity toward leukotriene B(4) and polyunsaturated fatty acids in the human hepatoblastoma cell line, HepG2, and human lung adenocarcinoma cell line, A549.

The addition of glucose to the culture medium of HepG2 or A549 cells for 22 h caused a dose-dependent increase in leukotriene B(4) omega-hydroxylation activity in the homogenate. The addition of genistein to the culture medium of HepG2 or A549 cells for 22 h caused a dose-dependent decrease in the activity, although the number of living cells was not influenced by the addition of genistein. The inhibition by genistein was reversed by removal of genistein from the culture medium in 22 h. The specific leukotriene B(4) omega-hydroxylation activity was high in the nuclear envelope fraction of HepG2 or A549 cells, and a large portion of the activity was concentrated in the nuclear envelope fraction. In the nuclear envelope fraction, leukotriene B(4) omega-hydroxylation activity was accompanied by high polyunsaturated fatty acid omega-hydroxylation activity. The apparent K(m) values for arachidonic acid and leukotriene B(4) in the fractions of HepG2 or A549 cells were 25 and 50 microM, or 22 and 66 microM, respectively. The V(max) values were 222 and 104 pmol/min/mg protein, or 175 and 370 pmol/min/mg protein, respectively. NADPH-dependent omega-hydroxylation of LTB(4) in the nuclear envelope fraction of HepG2 or A549 cells was strongly inhibited by metyrapone and CO. The expression of cytochrome P450 4F2 mRNAs was detected in HepG2 and A549 cells, and thus the arachidonic acid and leukotriene B(4) omega-hydroxylation activities in the nuclear envelope fractions of HepG2 and A549 cells are likely due to cytochrome P450 4F2.

Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons.

Mutations of presenilin 1 (PS1) causing Alzheimer's disease selectively increase the secretion of the amyloidogenic betaA4(1-42), whereas knocking out the gene results in decreased production of both betaA4(1-40) and (1-42) amyloid peptides (De Strooper et al. 1998). Therefore, PS1 function is closely linked to the gamma-secretase processing of the amyloid precursor protein (APP). Given the ongoing controversy on the subcellular localization of PS1, it remains unclear at what level of the secretory and endocytic pathways PS1 exerts its activity on APP and on the APP carboxy-terminal fragments that are the direct substrates for gamma-secretase. Therefore, we have reinvestigated the subcellular localization of endogenously expressed PS1 in neurons in vitro and in vivo using confocal microscopy and fine-tuned subcellular fractionation. We show that uncleaved PS1 holoprotein is recovered in the nuclear envelope fraction, whereas the cleaved PS fragments are found mainly in post-ER membranes including the intermediate compartment (IC). PS1 is concentrated in discrete sec23p- and p58/ERGIC-53-positive patches, suggesting its localization in subdomains involved in ER export. PS1 is not found to significant amounts beyond the cis-Golgi. Surprisingly, we found that APP carboxy-terminal fragments also coenrich in the pre-Golgi membrane fractions, consistent with the idea that these fragments are the real substrates for gamma-secretase. Functional evidence that PS1 exerts its effects on gamma-secretase processing of APP in the ER/IC was obtained using a series of APP trafficking mutants. These mutants were investigated in hippocampal neurons derived from transgenic mice expressing PS1wt or PS1 containing clinical mutations (PS1(M146L) and PS1(L286V)) at physiologically relevant levels. We demonstrate that the APP-London and PS1 mutations have additive effects on the increased secretion of betaA4(1-42) relative to betaA4(1-40), indicating that both mutations operate independently. Overall, our data clearly establish that PS1 controls gamma(42)-secretase activity in pre-Golgi compartments. We discuss models that reconcile this conclusion with the effects of PS1 deficiency on the generation of betaA4(1-40) peptide in the late biosynthetic and endocytic pathways.

Demonstration of Equilibrative Nucleoside Transporters (hENT1 and hENT2) in Nuclear Envelopes of Cultured Human Choriocarcinoma (BeWo) Cells by Functional Reconstitution in Proteoliposomes

The equilibrative nucleoside transporters (ENTs) are a newly recognized family of membrane proteins of which hENT1 is the nitrobenzylmercaptopurine ribonucleoside (NBMPR)-sensitive (es) and hENT2 the NBMPR-insensitive (ei) transporter of human cells. BeWo cells exhibit large numbers (>10(7)/cell) of NBMPR-binding sites and high es and ei nucleoside transport activities relative to other cell types. In this work, we have demonstrated that proliferating BeWo cells possess (i) mRNA encoding hENT1 and hENT2 and (ii) hENT1-specific immunoepitopes. We examined NBMPR binding and its inhibition of uridine transport in various BeWo membrane fractions and proteoliposomes derived therefrom to determine if NBMPR binding to intracellular membranes represented interaction with functional es transporters. Unfractionated membranes and fractions enriched 5-fold in plasma membranes relative to postnuclear supernatants exhibited high NBMPR binding activity. Intact nuclei and nuclear envelopes also exhibited abundant quantities of NBMPR-binding sites with affinities similar to those of enriched plasma membranes (Kd = 0.4-0.9 nM). When proteoliposomes were made from octyl glucoside-solubilized membranes, high affinity NBMPR-binding sites were not only observed in crude membrane preparations and plasma membrane-enriched fractions but also in nuclear envelope fractions. Proteoliposomes prepared from either unfractionated membranes or nuclear envelopes exhibited both hENT1-mediated (82-85%) and hENT2-mediated (15-18%) transport of [3H]uridine. These results provided evidence for the presence of functional es and ei transporters in nuclear membranes and endoplasmic reticulum, suggesting that hENT1 and hENT2 may function in the translocation of nucleosides between the cytosol and the luminal compartments of one or both of these membrane types.

Substrate specificities of neuronal nuclear acetyltransferases involved in the synthesis of platelet-activating factor: Differences in the use of 1-alkyl and 1-acyl lysophospholipid acceptors

The selectivity of alkylglycerophosphate (AGP) acetyltransferase and lyso-platelet-activating factor (lyso-PAF) acetyltransferase was studied in neuronal nuclei isolated from cerebral cortices of 15-day-old rabbits. Specifically, 1-alkyl and 1-acyl analogues were compared as acceptors in these acetylation reactions. A number of observations supported one nuclear activity in the acetylation of AGP and lyso-PA. Lyso-PA was a competitive substrate for AGP, K m values for AGP and lyso-PA were similar, as were acetylation rates measured at individual AGP or lyso-PA concentrations, and the acetylation of both substrates was unaffected by preincubations with protein phosphatase 1 (PP-1). In contrast, there were a number of differences seen in the acetylation of lyso-PAF and lyso-PC. The kinetics for lyso-PC acetylation (as a function of lyso-PC concentration) were not hyperbolic, and lyso-PC was not a competitive substrate for the acetylation of lyso-PAF. Unlike acetylation rates with lyso-PAF, lyso-PC acetylation was not reduced by preincubations with PP-1, and was less susceptible to inhibition particularly at high levels of free fatty acid. In addition, rates of acetylation of lyso-PC were selectively increased by the presence of lyso-PA. When neuronal nuclear envelope fractions (NE) were prepared from N 1, the specific acetylation activity with lyso-PAF was significantly lower in NE, while the activities for lyso-PC were comparable in NE and the parent N 1 fraction. The results with the acetylation of lyso-PC and lyso-PAF suggest that the lyso-PC acetyltransferase may be in a uniquely sequestered state within the neuronal nucleus. This could explain the smaller inhibition of lyso-PC acetylation by free fatty acid, the maintenance of lyso-PC acetylation during PP-1 preincubations, the non-hyperbolic response to lyso-PC concentrations and the selective preservation of lyso-PC acetylation during NE isolation. This protected status could result from a more internal location for this acetyltransferase within the membranes of the nuclear envelope, or possibly an association of the enzyme with the nuclear matrix that is disrupted with the exposure of N 1 to lyso-PA.

P53 Is Associated with the Nuclear Envelope in Mouse Testis

p53 has been postulated to play a role in meiosis as well as in the regulation of germ cell numbers by apoptosis. This study investigated the subcellular localization of p53 in the testis, including conditions known to induce germ cell apoptosis. Western blot analysis showed that p53 was enriched in the nuclear envelope fraction, and confocal microscopy confirmed that p53 was associated with the nuclear envelope of germ cells. Exposure of the testis to heat stress induced translocation of p53 into the nucleus. Nuclear envelope binding provides an optimal site for rapid entry of p53 into the nucleus, where it may act as a DNA-binding protein to induce apoptosis or cell cycle arrest in response to appropriate stimuli. The nuclear envelope sequestration of p53 also provides a framework to understand how mitosis and meiosis in the testis may proceed despite high intracellular concentration of p53.

Neuronal nuclear acetyltransferases involved in the synthesis of platelet-activating factor are located in the nuclear envelope and show differential losses in activity

Neuronal nuclear fraction N 1 was isolated from cerebral cortices of 15-day-old rabbits, and nuclear subfractions prepared, in order to study the location of nuclear lyso platelet-activating factor (lyso-PAF) acetyltransferase and alkylglycerophosphate (AGP) acetyltransferase, and factors that affect the loss of these two nuclear activities. Subfractionation of prelabelled N 1 indicated that the nuclear envelope had the highest percentage of the radioactive acetylated products alkylacetylglycerophosphate (AAGP) and PAF, and the distribution of these phospholipids reflected phospholipid distributions in the nuclear subfractions. The majority (95%) of radioactive AAGP and PAF was also recovered in Triton X-100 extracts of prelabelled nuclei, suggesting that these acetylated lipids are located in nuclear membranes rather than in the nuclear matrix/chromatin. Of the nuclear subfractions, the envelope had the highest AGP and lyso-PAF acetyltransferase specific activities which were close to corresponding values seen in the parent N 1 fraction. Thus the nuclear AGP and lyso-PAF acetyltransferases were principally localized to the nuclear membranes. Differentials in activity loss were seen for the two acetyltransferase activities. In the nuclear envelope fractions, the lyso-PAF acetyltransferase was the more susceptible to oxidation reactions which could be reversed or blocked by the use of reducing agents. In preincubations, N 1 showed greater losses in lyso-PAF acetyltransferase activity than in AGP acetyltransferase activity, losses which were not attributable to oxidation. Addition of cytosolic fraction S 3 to preincubations promoted losses for each acetyltransferase in N 1, and gave evidence for cytosolic and endogenous nuclear contributions to the activity loss. Addition of okadaic acid to the preincubations did not prevent the decline of either acetyltransferase in intact nuclei, but did diminish the loss of nuclear lyso-PAF acetyltransferase activity promoted by S 3 addition, and also blocked the loss of this acetyltransferase seen in preincubations of isolated nuclear envelopes. This suggests that nuclear lyso-PAF acetyltransferase is susceptible to okadaic acid-sensitive nuclear and cytosolic protein phosphatase activities, while AGP acetyltransferase may lose activity by the action of other phosphatases or by other mechanisms within the nucleus.

Characterization of p92, karyopherin beta, co-purified with N-acetylglucosamine-bearing nucleoporins from rat liver nuclear envelopes.

A 92k protein (p92) was purified from the wheat germ agglutinin-Sepharose (WGA-Sepharose) bound fraction of a rat liver nuclear envelope salt-extract by DEAE-5PW and hydroxyapatite HPLCs. Partial amino acid sequence analysis of p92 revealed that it is karyopherin beta, which was found recently in the cytosolic fraction. It was shown using anti-p92 antiserum that the protein is present in the nuclear envelope and cytosolic fractions, in almost the same amounts, but not in other subcellular fractions of rat liver. p92 bound to N-acetylglucosamine bearing nucleoporins (GNPs) on WGA-Sepharose, but not directly to WGA. The amount of p92 found in the rat liver nuclear envelope fraction corresponded to about 10% of the nuclear pore complex in mass, and to as much as 140 mol of p92 per mol of nuclear pore complex. Hydrodynamic analysis of the purified p92 suggested that the molecule is present as a monomer and that it is a rod-shaped molecule. The interaction of p92 and GNPs seemed to be hydrophobic and ionic. Based on these results, the participation of nuclear envelope p92 in protein nuclear transport is discussed.

Subcellular Distribution and Phosphorylation of the Nuclear Localization Signal Binding Protein, NBP60

We previously purified a nuclear localization signal binding protein, NBP60, from rat liver (1993,J. Biochem.113, 308–313). In this study, the subcellular localization of NBP60 was examined using anti-NBP60. Most NBP60 was found to be localized in the nuclear envelope fraction of rat liver obtained on cell fractionation followed by immunoblotting. Staining of the nuclei of cultured cells by the antibody was observed on immunofluorescence microscopy. NBP60 was widely detected in rat nuclear fractions prepared from other tissues and also in nuclei of cultured cells derived from other species. It was shown by immunoelectron microscopy that most NBP60 is present in the nuclear envelope and at least some of that is present on nuclear pore complexes. Although NBP60 was localized in the nuclear envelope in interphase cells, it diffused into the cytoplasm in the mitotic phase. The purified NBP60 was highly phosphorylated by a cdc2 mitotic kinase, whereas nuclear pore proteins p144, p62, p60, and p54 were not phosphorylated by the kinase directly. NBP60 was also phosphorylated by protein kinase A, calmodulin-dependent protein kinase II, and casein kinase II. The phosphorylation of NBP60 by cdc2 kinase and/or the other kinases may be related to the change in the protein's location during the mitotic phase.

Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei.

Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

Deletion of the regulatory domain of protein kinase C alpha exposes regions in the hinge and catalytic domains that mediate nuclear targeting.

Members of the protein kinase C (PKC) family are characterized by an NH2-terminal regulatory domain containing binding sites for calcium, phosphatidylserine, and diacylglycerol (or tumor-promoting phorbol esters), a small central hinge region and a COOH-terminal catalytic domain. We have constructed fusion proteins in which the regulatory domain of PKC alpha was removed and replaced by a 19-amino acid leader sequence containing a myristoylation consensus or by the same sequence in which the amino-terminal glycine was changed to alanine to prevent myristoylation. The goal was to generate constitutively active mutants of PKC that were either membrane bound, due to their myristoylation, or cytoplasmic. Western blotting of fractions from COS cells transfected with plasmids encoding wild-type and mutant proteins revealed that PKC alpha resided entirely in a Triton X-100 soluble (TS) fraction, whereas both the myristoylated and nonmyristoylated mutants were associated primarily with the nuclear envelope fraction. A similar mutant that lacked the 19 amino acid leader sequence was also found almost entirely in the nuclear envelope, as was a truncation mutant containing only the regulatory domain, hinge region, and a small portion of the catalytic domain. However, an additional truncation mutant consisting of only the regulatory domain plus the first one-third of the hinge region was almost entirely in the TS fraction. A nonmyristoylated fusion protein containing only the catalytic domain was also found in the nuclear envelope. Immunostaining of cells transfected with these constructs revealed that both the myristoylated and nonmyristoylated mutants were localized in nuclei, whereas wild-type PKC alpha was primarily cytoplasmic and perinuclear. Phorbol dibutyrate treatment of PKC alpha-transfected cells resulted in increased perinuclear and nuclear staining. The results are consistent with a model in which activation of PKC, by phorbol esters or by deletion of the regulatory domain, exposes regions in the hinge and catalytic domains that interact with a PKC "receptor" present in the nuclear envelope, and may explain the ability of wild-type PKC to be translocated to the nucleus under certain conditions.