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R A Laskey

Publications and source records attributed to R A Laskey.

At least 37 records · Page 2Linked to original sources
PubMed1997

Molecular and cellular characterization of CRP1, a Drosophila chromatin decondensation protein.

CRP1, a Drosophila nuclear protein that can catalyze decondensation of demembranated Xenopus sperm chromatin was cloned and its primary structure was deduced from cDNA sequence. Alignment of deduced amino acid sequence with published sequences of other proteins revealed strong homologies to Xenopus nucleoplasmin and NO38. CRP1 is encoded by one or several closely related genes found at a single locus, position 99A on the right arm of chromosome 3. CRP1 mRNA is expressed throughout Drosophila development; it is highest during oogenesis and early embryogenesis. mRNA levels correlate closely with levels of protein expression measured previously. Results of chemical crosslinking indicate that CRP1 is either tetrameric or pentameric; similar ambiguity was revealed by direct visualization using scanning transmission electron microscopy. Consistent with previously published results, parallel crosslinking studies of Xenopus nucleoplasmin suggested a pentameric structure. Scanning transmission electron microscopic examination after negative staining revealed that CRP1 and Xenopus nucleoplasmin are morphologically similar. CRP1 is able to substitute for nucleoplasmin in Xenopus egg extract-mediated sperm chromatin decondensation. In vitro, CRP1-induced decondensation is accompanied by direct binding of CRP1 to chromatin.

Amino Acid Sequence↗
PubMed1996

Regulatory roles of the nuclear envelope.

Roles of the nuclear envelope are considered in the regulation of nuclear protein import, ribonucleoprotein export, and coupling of DNA replication to the cell cycle. First, evidence is discussed that indicates that neutral and acidic amino acids can be important in nuclear localization signals as well as the widely acknowledged basic amino acids. Second, the recognition of nuclear localization signals by their receptor "importin" is discussed, focusing on the different roles of the two subunits of importin. Third, a role for the alpha subunit of importin in RNP export is considered together with the question of how the direction of traffic through nuclear pores is determined. The final part of this article considers evidence that the nuclear membrane prevents reinitiation of DNA replication in Xenopus eggs, by excluding a "licensing factor" that is essential for DNA replication. Replication licensing in Xenopus appears to involve several proteins including the MCM (minichromosome maintenance) complex and ORC, the origin recognition complex, which must bind before the MCM complex can bind to chromatin.

Amino Acid Sequence↗
PubMed1996

The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin.

BACKGROUND: The origin recognition complex (ORC) and the minichromosome maintenance (MCM) protein complex were initially discovered in yeast and shown to be essential for DNA replication. Homologues of ORC and MCM proteins exist in higher eukaryotes, including Xenopus. The Xenopus MCM proteins and the Xenopus homologues of Saccharomyces cerevisiae Orc 1p and Orc2p (XOrc1 and XOrc2) have recently been shown to be essential for DNA replication. Here, we describe the different but interdependent functions of the ORC and MCM complexes in DNA replication in Xenopus egg extracts. RESULTS: The XOrc1 and XOrc2 proteins are present in the same multiprotein complex in Xenopus egg extracts. Immunodepletion of ORC inhibits DNA replication of Xenopus sperm nuclei. Mixing MCM-depleted and ORC-depleted extracts restores replication capacity. ORC does not co-localize with sites of DNA replication during elongation. However, at initiation the two staining patterns overlap. In contrast to MCMs, which are displaced from chromatin during S phase, XOrc1 and XOrc2 are nuclear chromatin-bound proteins throughout interphase and move to the cytoplasm in mitosis. Permeable HeLa G1- and G2-phase nuclei can replicate in ORC-depleted extract, consistent with the presence of chromatin-bound ORC in both pre-replicative and post-replicative nuclei. Interestingly, the binding of ORC to chromatin does not require the presence of MCMs; however, the binding of MCM proteins to chromatin is dependent on the presence of ORC. CONCLUSIONS: The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication. Xenopus ORC is bound to chromatin throughout interphase but, in contrast to S. cerevisiae ORC, it appears to be, at least partly, displaced from chromatin during mitosis. The binding of MCM proteins requires the presence of ORC. Thus, the assembly of replication-competent chromatin involves the sequential binding of ORC and MCMs to DNA.

Animals↗
PubMed1996

Importin provides a link between nuclear protein import and U snRNA export.

Importin-alpha mediates nuclear protein import by binding nuclear localization signals and importin-beta. We find approximately 30% of SRP1p, the yeast importin-alpha, in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC). Similarly, a large fraction of Xenopus CBC is associated with importin-alpha in the nucleus. CBC promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm. The CBC-importin-alpha complex binds specifically to capped RNA, suggesting that CBC might shuttle while bound to importin-alpha. Strikingly, importin-beta binding displaces the RNA from the CBC-importin-alpha complex. Thus, the commitment of CBC for nuclear reentry triggers the release of the export substrate into the cytoplasm. We provide evidence for a mechanism that ensures that importin-mediated RNA release is a specifically cytoplasmic event.

Amino Acid Sequence↗
PubMed1996

XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.

Animals↗
PubMed1996

Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids.

Nuclear proteins contain information within their primary structures which causes them to accumulate selectively in the nucleus [1,2] by associating with the cytosolic receptor importin [3]. The alpha subunit of importin binds the nuclear localization signal (NLS), and the beta subunit docks at the nuclear pore complex. The NLS of the simian virus 40 large T-antigen (SV40 T-ag) is a single cluster of basic amino acids (PKKKRKV132; single-letter code, the basic amino acids are shown in bold; [4,5]), whereas the NLS of nucleoplasmin is bipartite. The nucleoplasmin NLS requires two essential clusters of basic amino acids, separated by a mutation-tolerant spacer (KRPAATKKAGQAKKKK171; [6] [7]). A SwissProt database search shows that more than 50% of nuclear proteins contain a match to this consensus, and many NLSs have since been found to conform to this type of motif in yeast, plants and animals [8-10]. A different NLS (PAAKRVKLD) has been reported in the oncoprotein c-Myc, but it has received little attention because, unlike other known NLSs, only three of nine residues are basic [11], and one residue is even acidic. Here, we report that constructs containing an inactive basic cluster downstream of the bipartite signal of nucleoplasmin can be directed to the nucleus by flanking them with specific neutral and acidic residues taken from the signal reported for c-Myc. Nuclear targeting by the single cluster KKKK is dependent on it being preceded by PAA and is stimulated if it is followed by the dipeptide LD. The relative positions of these elements are crucial to the function of these NLSs. All regions of the unconventional signal of c-Myc are functionally important. Contrary to conventional views, neutral and even acidic amino acids can play crucial roles in NLSs.

Amino Acid Sequence↗
PubMed1996

A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus.

The complex of importin-alpha and -beta is essential for nuclear protein import. It binds the import substrate in the cytosol, and the resulting trimeric complex moves through the nuclear pores, probably as a single entity. Importin-alpha provides the nuclear localization signal binding site, importin-beta the site of initial docking to the pore. Here we show that the conserved, basic N-terminus of importin-alpha is sufficient for importin-beta binding and essential for protein import. The fusion product of this 41 amino acid domain to a heterologous protein if transported into the nucleus in the same way as full-length importin-alpha itself. Transport is dependent on importin-beta but competed by importin-alpha. As no additional part of importin-alpha is needed for translocation, the movement which drives the import substrate complex into the nucleus appears to be generated between importin-beta and structures of the nuclear pore. The domain that binds to importin-beta appears to confer import only, but not re-export out of the nucleus, suggesting that the return of importin-alpha into the cytoplasm is not a simple reversal of its entry.

Amino Acid Sequence↗
PubMed1996

Hyperphosphorylation of nucleoplasmin facilitates Xenopus sperm decondensation at fertilization.

Previous studies showed that the nuclear phosphoprotein nucleoplasmin performs the first stage of chromatin decondensation of Xenopus sperm at fertilization. It binds and removes sperm basic proteins replacing them with histones. We now show that this activity depends upon the massive hyperphosphorylation of nucleoplasmin that occurs when oocytes mature into eggs. Egg extracts or purified hyperphosphorylated egg nucleoplasmin decondense sperm chromatin and remove sperm basic proteins much faster than oocyte extracts or hypophosphorylated oocyte nucleoplasmin. Furthermore, dephosphorylation of egg nucleoplasmin slows sperm decondensation and prevents basic protein removal from sperm chromatin. We conclude that hyperphosphorylation of nucleoplasmin is used to modulate the rapid changes in chromatin structure that accompany early development in Xenopus.

Animals↗
PubMed1996

Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication.

Members of the Mcm-protein family have recently been shown to be involved in restricting DNA replication to a single cycle in Xenopus laevis egg extracts. In this study, we extended these observations to human somatic cells and analysed the localisation of the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 in replicating HeLa cell nuclei. These Mcm-proteins are entirely nuclear in interphase cells and apparently exist in two populations: a nucleosolic population, and a population bound to a nuclear structure, most likely chromatin. The bound population is detected throughout the nucleus in late G1 and early S, and at discrete subnuclear sites following further progression of S-phase. We use high resolution confocal microscopy to determine the subnuclear sites of chromatin-bound Mcm proteins in comparison to the sites of replicating DNA. Importantly, hCdc21, hCdc46 and P1Mcm3 do not colocalise with replication foci, instead these proteins appear to coincide with subnuclear sites of unreplicated chromatin. During progression of S-phase hCdc21, hCdc46 and P1Mcm3 are displaced from their site on chromatin at the time when this site is replicated. Consequently, early replicating sites do not contain bound hCdc21, hCdc46 or P1Mcm3 during later stages of S-phase. Furthermore, G2 nuclei and condensed chromatin in mitotic cells do not contain bound hCdc21, hCdc46 or P1Mcm3. Thus, the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 are not concentrated at sites of DNA replication. Instead, they appear to be present only on unreplicated chromatin and are displaced from replicating chromatin, consistent with a role in monitoring unreplicated chromatin and ensuring only a single round of DNA replication per cell cycle.

Animals↗
PubMed1995

The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts.

BACKGROUND: A complex of MCM proteins is implicated in ensuring that DNA replicates only once in each cell cycle, by 'replication licensing'. The nuclear membrane is also implicated in replication licensing, but the relationship between the MCM proteins and the nuclear membrane is unclear. Here, we investigate the relationship between XMCM3 (a component of the Xenopus MCM complex), nuclear envelope permeability and the initiation of DNA replication once per cell cycle. RESULTS: Our results show that the nuclear envelope does not prevent the entry of XMCM3 into the nucleus, but that it does prevent the binding of XMCM3 to chromatin. We have also identified another component of the Xenopus MCM complex as a homologue of the Schizosaccharomyces pombe protein Cdc21. XMCM3 does not preferentially co-localize with sites of DNA replication. Instead, it is almost uniformly distributed on chromatin and is suddenly lost during replication. XMCM3 crosses intact nuclear membranes of G2-phase HeLa cells but cannot then bind to chromatin. Permeabilization of the nuclear envelope allows the binding of XMCM3 to G2-phase chromatin. We have therefore resolved replication licensing into two stages. The first requires the entry of a cytosolic 'loading factor' that is excluded by the nuclear membrane; subsequently, MCM3 can bind to chromatin in the presence or absence of a nuclear membrane, but only if the loading factor has gained access in the absence of the membrane. CONCLUSIONS: The Xenopus MCM complex contains homologues of yeast MCM2, MCM3, MCM5 and Cdc21 proteins. XMCM3 is displaced from chromatin during replication. The nuclear envelope allows entry of XMCM3 into the nucleus, but regulates its binding to chromatin; binding requires a loading factor which cannot cross the nuclear envelope. Based on these results we present a two-stage model for replication licensing.

Amino Acid Sequence↗
PubMed1995

Distinct functions for the two importin subunits in nuclear protein import.

The import of nuclear proteins proceeds through the nuclear pore complex and requires nuclear localization signals (NLSs), energy and soluble factors, namely importin-alpha (M(r) 60K), importin-beta (90K) and Ran. Importin-alpha is primarily responsible for NLS recognition and is a member of a protein family that includes the essential yeast nuclear pore protein SRP1p (ref. 16). As the first event, the complex of importin-alpha and importin-beta binds the import substrate in the cytosol. Here we show that this nuclear pore targeting complex initially docks as a single entity to the nuclear pore via importin-beta. Then the energy-dependent, Ran-mediated translocation through the pore results in the accumulation of import substrate and importin-alpha in the nucleus. In contrast, importin-beta accumulates at the nuclear envelope, but not in the nucleoplasm. Immunoelectron microscopy detects importin-beta on both sides of the nuclear pore. This suggests that the nuclear pore targeting complex might move as a single entity from its initial docking site through the central part of the nuclear pore before it disassembles on the nucleoplasmic side.

Adenosine Triphosphate↗
PubMed1995

MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells.

An intact nuclear membrane restricts DNA replication to only one round in each cell cycle, apparently by excluding an essential replication-licensing factor throughout interphase. A family of related yeast replication proteins, MCM2, 3 and 5 (also called, after cell-division cycle, CDC46), resemble licensing factor, entering the nucleus only during mitosis. We have cloned a Xenopus homologue of MCM3 (XMCM3) and raised antibodies against expressed protein. Immunodepletion of Xenopus egg extracts removes a complex of MCM2, 3 and 5 homologues and inhibits replication of Xenopus sperm nuclei or permeable G2 HeLa nuclei. However, G1 HeLa nuclei still replicate efficiently. Mock-depleted extracts replicate all three templates. XMCM3 accumulates in nuclei before replication but anti-XMCM3 staining decreases during replication. These results can explain why replicated nuclei are unable to reinitiate replication in a single cell cycle.

Amino Acid Sequence↗
PubMed1995

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope.

BACKGROUND: Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide. RESULTS: We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. CONCLUSIONS: Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

Amino Acid Sequence↗
PubMed1994

Isolation of a protein that is essential for the first step of nuclear protein import.

We have purified a cytosolic protein from Xenopus eggs that is essential for selective protein import into the cell nucleus. The purified protein, named importin, promotes signal-dependent binding of karyophilic proteins to the nuclear envelope. We have cloned, sequenced, and expressed a corresponding cDNA. Importin shows 44% sequence identity with SRP1p, a protein associated with the yeast nuclear pore complex. Complete, signal-dependent import into HeLa nuclei can be reconstituted by combining importin purified from Xenopus eggs or expressed in E. coli with Ran/TC4. Evidence for additional stimulatory factors is provided.

Adenosine Triphosphate↗
PubMed1993

The role of nucleoplasmin in chromatin assembly and disassembly.

Nucleoplasmin is the most abundant nuclear protein in Xenopus oocytes and eggs. The term 'molecular chaperone' was coined to describe its role in the assembly of the nucleosome subunits of chromatin. Although histones and DNA can self-assemble into nucleosomes, nucleoplasmin can facilitate this process in vitro by competing against non-specific charge interactions. In vivo nucleoplasmin binds histones H2A and H2B and transfers them to DNA. Another acidic nuclear protein, N1, binds and transfers histones H3 and H4. Nucleoplasmin has at least one other role in modulating chromatin structure in Xenopus eggs. It is required for the first stage of sperm chromatin decondensation. It binds and removes sperm basic proteins and replaces them by histones H2A and H2B, again forming nucleosomes, and resulting in decondensation of the compacted sperm chromatin. In addition we propose that the properties of the nuclear localization signal of nucleoplasmin can be explained by a model in which heat shock cognate protein hsc70 has a chaperone role in signal presentation during nuclear transport.

Amino Acid Sequence↗