Ronald A. Laskey

Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: Identification of a class of bipartite nuclear targeting sequence

Point mutagenesis of the nuclear targeting sequence of nucleoplasmin has identified two interdependent basic domains. These are separated by 10 intervening spacer amino acids that tolerate point mutations and some insertions. Amino acids in both basic domains are required for nuclear targeting, and the transport defect of a mutation in one domain is amplified by a simultaneous mutation in the other. Therefore, these basic domains are interdependent. A strikingly similar motif of two clusters of basic residues is seen in the nuclear targeting sequence of Xenopus N1. It is also conserved in the related nucleolar protein NO38. Several other short sequences known to be necessary for nuclear targeting fall within a similar motif.

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.

Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.

We demonstrate that cell-free extracts prepared from activated eggs of X. laevis by a method similar to that of Lohka and Masui initiate and complete semiconservative DNA replication of sperm nuclei and plasmid DNA. The efficiency of replication is comparable to that in the intact egg. Under optimal conditions 70%-100% of nuclei, and up to 38% of naked DNA molecules replicate completely. Genuine initiation of replication occurs rather than elongation of preformed primers or priming of irreversibly denatured templates. Rereplication of templates is observed under certain conditions. In addition to replicating DNA, these extracts also assemble nucleus-like structures from naked DNA.

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

BACKGROUNDnSelective 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.nnnRESULTSnWe 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.nnnCONCLUSIONSnImportin 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.

Nuclear protein migration involves two steps: Rapid binding at the nuclear envelope followed by slower translocation through nuclear pores

When injected into the cytoplasm of Vero cells, nucleoplasmin rapidly concentrates in a narrow layer around the nuclear envelope and then accumulates within the nucleus. Transport into the nucleus can be reversibly arrested at the perinuclear stage by metabolic inhibitors or by chilling. Nucleoplasmin-coated colloidal gold particles concentrate around the nuclear envelope of Vero cells or Xenopus oocytes, and by electron microscopy of oocytes appear to be associated with fibrils attached to nuclear pore complexes. Perinuclear accumulation is not observed for the nonmigrating nucleoplasmin core fragment or nonnuclear proteins. We propose two steps in nuclear migration of proteins: rapid binding around the nuclear envelope, possibly to pore-associated fibrils, followed by slower, energy-dependent translocation through nuclear pores.

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.

Cyclin/Cdk-dependent initiation of DNA replication in a human cell-free system.

We describe a cell-free system from HeLa cells that initiates DNA replication under cell cycle control. G1 but not G2 phase nuclei initiate replication when coincubated with S phase nuclei in cytosolic extracts from S phase but not from G1 or G2 phase HeLa cells. S phase nuclei or an S phase nuclear extract are required for the initiation of semiconservative DNA replication in G1 nuclei but not for elongation in S phase nuclei. S phase nuclear extract could be replaced by recombinant human cyclins A and E complexed to Cdk2 but not by Cdk2 alone or by human cyclin B1 complexed to Cdc2. In S phase cytosol, cyclins A/Cdk2 and E/Cdk2 triggered initiation synergistically.

Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin

At fertilization, sperm chromatin decondenses in two stages, which can be mimicked in extracts of Xenopus eggs. Rapid, limited decondensation is followed by slower, membrane-dependent decondensation and swelling. Nucleoplasmin, an acidic nuclear protein, occurs at high concentration in Xenopus eggs and has a histone-binding role in nucleosome assembly. Immunodepleting nucleoplasmin from egg extracts inhibits the initial rapid stage of sperm decondensation, and also the decondensation of myeloma nuclei, relative to controls of mock depletion and TFIIIA depletion. Readdition of purified nucleoplasmin recues depleted extracts. A physiological concentration of purified nucleoplasmin alone decondenses both sperm and myeloma nuclei. We conclude that nucleoplasmin is both necessary and sufficient for the first stage of sperm decondensation in Xenopus eggs.

Distinct roles for cyclins E and A during DNA replication complex assembly and activation

Initiation of DNA replication is regulated by cyclin-dependent protein kinase 2 (Cdk2) in association with two different regulatory subunits, cyclin A and cyclin E (reviewed in ref. 1). But why two different cyclins are required and why their order of activation is tightly regulated are unknown. Using a cell-free system for initiation of DNA replication that is based on G1 nuclei, G1 cytosol and recombinant proteins, we find that cyclins E and A have specialized roles during the transition from G0 to S phase. Cyclin E stimulates replication complex assembly by cooperating with Cdc6, to make G1 nuclei competent to replicate in vitro. Cyclin A has two separable functions: it activates DNA synthesis by replication complexes that are already assembled, and it inhibits the assembly of new complexes. Thus, cyclin E opens a window of opportunity for replication complex assembly that is closed by cyclin A. The dual functions of cyclin A ensure that the assembly phase (G1) ends before DNA synthesis (S) begins, thereby preventing re-initiation until the next cell cycle.

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.