The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen.

Abstract

Abstract. The carboxy-terminal tail of nucleoplasmin, which specifies entry into the cell nucleus, contains four short sequences that are similar to previously identified nuclear location sequences. We show that none of these is able to locate chicken muscle pyruvate kinase to the cell nucleus. Deletion analysis was used to determine the limits of a nuclear location sequence and indicated that a 14-amino acid segment (RPAATKKAGQAKKK) should function as a minimal nuclear location sequence. When tested directly, however, this sequence was unable to locate pyruvate kinase to the cell nucleus. Restoration of three amino acids of nucleoplasmin sequence at either end of this sequence generated sequences that were able to locate pyruvate kinase to the cell nucleus.The 14-amino acid proposed minimal nuclear location sequence is present in the functional sequences, AVKRPAATKKAGQAKKK, RPAATKKAG-QAKKKKLD, and the sequence AVKRPAATKKAG-QAKKKKLD, which has additional amino acids at both ends. The minimal sequence element is therefore necessary but not sufficient for transport into the cell nucleus. This unusual feature of the nucleoplasmin nuclear location sequence suggests ways in which it could interact with the nuclear transport mechanism. IN addition to its established role in chromatin assembly and histone storage in the Xenopus oocyte and egg (4,16,18,21), nucleoplasmin has emerged as the protein of choice for studies of protein transport into the cell nucleus (reviewed in reference 5). Investigations using this acidic, thermostable, pentameric protein provided the decisive evidence that a mechanism of uptake involving selective entry into the nucleus operates in oocytes (8).Nucleoplasmin has also been used to demonstrate that entry into the Xenopus oocyte nucleus occurs through the nuclear pore complex (9), to evaluate the transport properties of nuclei reconstituted in vitro, and to show that ATP is required for the transport process (28). Primary amino acid sequences specifying entry into the nucleus have been identified in a number of proteins (5,23,25,30). The sequence that has been studied in greatest molecular detail is the SV-40 large T antigen nuclear location sequence (3,11,14,15,19,20,33,37). The earlier studies in this series demonstrated that the amino acid sequence PKK~28KRKV is able to translocate chicken muscle pyruvate kinase to the cell nucleus (15).The chicken muscle pyruvate kinase expression vector system has also been used to map the polyoma virus large T nu-C. Dingwall's present address is Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, England.clear location sequences (32) and more recently to investigate the effect of protein context upon the function of the SV-40 large T antigen nuclear location sequence (33). Therefore we chose this system to identify the nuclear location sequence or sequences in the nucleoplasmin "tail" region since a large body of data exists against which we can interpret our results.We have sequenced nucleoplasmin and shown that the tail regi...

Landscape of nuclear transport receptor cargo specificity

“…This analysis recovered KR‐rich motifs that are part of the cNLS as the main motif of importin α cargos, and P‐rich motifs similar to the PY‐NLS as the main motif of transportin cargos (Lee et al , 2006), as expected (Table EV6), despite the limitation that DILIMOT is not designed to recover bipartite motifs. In both cases, the identified motifs are similar but not identical to the definitions proposed in previous literature (Kalderon et al , 1984; Dingwall et al , 1988; Lee et al , 2006), underlining that the respective signals exhibit a certain degree of plasticity. Surprisingly, this analysis also identified acidic stretches (DE‐rich motifs) to be significantly enriched in cargos of various importin β‐type NTRs but not, for example, transportins (Table EV6).…”

Molecular Cloning and Characterization of a Novel Mouse Macrophage Gene That Encodes a Nuclear Protein Comprising Polyglutamine Repeats and Interspersing Histidines

“…Transcription factors often carry clusters of positive net charge, and these clusters consisting of basic residues are often associated with DNA binding domains (23). The putative NLS (amino acids [17][18][19][20] is similar to that first identified in the large T-antigen of simian virus 40 (61,62) versus the bipartite NLS first identified in the nonnucleic acid binding protein nucleoplasmin (63,64). Residues 17-20 satisfy the requirements of a minimal NLS by having the tetrameric sequence K(R/K)X(R/K) in which X is K, R, P, V, or A but not N (47).…”

A GR-motif functions in nuclear accumulation of the large FGF-2 isoforms and interferes with mitogenic signalling

“…To identify and study potential nuclear targeting signals present in the chicken FGF-2 isoforms, di erent parts were fused to pyruvate kinase (PK), a cytoplasmic protein commonly used to study NLS function (Dingwall et al, 1988). The sub-cellular distribution of the di erent fusion proteins was analysed in NIH3T3 ®broblasts using anti-PK antibodies (Figure 1; for details see Materials and methods).…”

“…The parental vectors encoding the chicken PK gene were previously described and permit carboxy-terminal fusions (Dingwall et al, 1988; kindly provided by C Dingwall). The PK-LLM construct was generated by fusing the chicken FGF-2 ORF (Zuniga Meija Borja et al, 1993) in-frame to PK.…”

“…Pyruvate kinase fusion proteins were detected using a polyclonal antiserum against chicken pyruvate kinase (Dingwall et al, 1988; kindly provided by C Dingwall). FGF-2 isoforms were identi®ed using a nity puri®ed polyclonal FGF-2 antibodies (Dono and Zeller, 1994).…”