Antibodies against the SV40 large T antigen nuclear localization sequence (original) (raw)
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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 a subunit of importin binds the nuclear localization signal (NLS), and the b subunit docks at the nuclear pore complex. The NLS of the simian virus 40 large T-antigen (SV40 Tag) is a single cluster of basic amino acids (PKKKRKV 132 ; 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 (KRPAATKKAGQAKKKK 171 ; [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 oncopro-tein 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 nucleo-plasmin 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.
Biochemical Journal, 2003
The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser 112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-α (Impα), we co-crystallized non-autoinhibited Impα with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impα. We confirm the important role of flanking sequences using binding assays. Un-expectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impα. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impα, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impα.
Analysis of conserved binding proteins for nuclear localization sequences
Journal of cell science, 1993
Correct targeting of nuclear proteins is mediated by nuclear localization sequences (NLS) which permit specific binding to the nucleus and subsequent translocation across the nuclear envelope via the nuclear pore complex. It is proposed that nuclear import is facilitated by NLS-receptors which reside in the cytoplasm and at the nuclear pore. These NLS-receptors could facilitate an early step of nuclear protein import, i.e. targeting and binding of nuclear proteins at the nuclear pore. We have generated anti-idiotype antibodies against the SV40 T-antigen nuclear localization sequence that allowed us to study NLS-binding proteins in a variety of different organisms. Proteins of similar size are recognized by these antibodies in yeast, Drosophila, rat and human cells. Cytological analysis indicates that the NLS-binding proteins reside in part at nuclear pores. One of the proteins recognized by anti-idiotype antibodies is identical to a previously identified NLS-binding protein. Using i...
Characterization of molecules involved in protein translocation using a specific antibody
The Journal of Cell Biology, 1982
The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol . 87:503-508) . Rabbit antibodies raised against this peptide were used to further characterize the membrane-bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived . The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system . Moreover, immunoprecipitation of elastase extracts alkylated with [t4 C] N-ethylmaleimide selected a single species of mol wt 60,000 .
Journal of Molecular Biology, 2000
Importin-a is the nuclear import receptor that recognizes cargo proteins which contain classical monopartite and bipartite nuclear localization sequences (NLSs), and facilitates their transport into the nucleus. To determine the structural basis of the recognition of the two classes of NLSs by mammalian importin-a, we co-crystallized an N-terminally truncated mouse receptor protein with peptides corresponding to the monopartite NLS from the simian virus 40 (SV40) large T-antigen, and the bipartite NLS from nucleoplasmin. We show that the monopartite SV40 large T-antigen NLS binds to two binding sites on the receptor, similar to what was observed in yeast importin-a. The nucleoplasmin NLS-importin-a complex shows, for the ®rst time, the mode of binding of bipartite NLSs to the receptor. The two basic clusters in the NLS occupy the two binding sites used by the monopartite NLS, while the sequence linking the two basic clusters is poorly ordered, consistent with its tolerance to mutations. The structures explain the structural basis for binding of diverse NLSs to the sole receptor protein. Abbreviations used: Arm repeats, armadillo repeats; Impa(70-529), importin-a lacking the N-terminal 69 residues; NLS, nuclear localization sequence; NPC, nuclear pore complex; SV40, simian virus 40; SV40 NLS, peptide 126 PKKKRKV 132 corresponding to the NLS of the SV40 large T-antigen; nucleoplasmin NLS, peptide 155 KRPAATKKAGQAKKKK 170 corresponding to the NLS of nucleoplasmin.
Molecular and cellular biology, 1988
A previous study demonstrated the ability of a synthetic peptide homologous to the simian virus 40 T-antigen nuclear transport signal to induce the nuclear transport of carrier proteins and the dependence of peptide-induced transport on a positive charge at the lysine corresponding to amino acid 128 of T antigen. In this investigation synthetic peptides were utilized to examine the effect on transport of amino acid substitutions within the T-antigen nuclear transport signal. Nuclear transport was evaluated by immunofluorescence after microinjection of protein-peptide conjugates into the cytoplasm of mammalian cells. Substitution of other basic amino acids at position 128 revealed a hierarchy for nuclear transport. The rate of nuclear transport was most rapid when a lysine was at position 128 followed in descending order by arginine, D-lysine, ornithine, and p-aminophenylalanine. Peptide-induced nuclear transport was dependent upon a positively charged amino acid at positions 128 and...
Experimental Cell Research, 1975
A number of observations suggest there are some non-histone proteins unique to constitutive heterochromatin which are responsible for some of its properties such as interphase condensation, C-banding, frequent decreased quinacrine fluorescence, ectopic pairing, and ability to cause position effect. To investigate this, mouse nuclei were disrupted by sonication and separated by differential centrifugation into heterochromatin+nucleoli, heterochromatin, intermediate, euchromatin pellet and euchromatin supernatant fractions. The non-histone proteins of these fractions were examined by SDS slab gel electrophoresis and compared with the proteins of the cytoplasm, nucleoplasm, cytoplasmic ribosomes, nuclear membrane, and to tubulin, actin, myosin and other muscle proteins. There were a number of non-histone proteins that were unique to or present in greater quantity in the heterochromatin+nucleoli fraction than in the euchromatin. Some of the chromatin non-histone proteins co-migrated with muscle proteins by SDS gel electrophoresis.
Cell, 1986
A system was developed for the analysis of protein transport to the nucleus. Carrier proteins cross-linked to synthetic peptides were microinjected into the cytoplasm of mammalian cells, and protein transport was evaluated by immunofluorescence staining of fixed cells. A Wmer synthetic peptide containing seven ammo acids homologous to SV40 T antigen was capable of inducing nuclear transport, but no transport was observed when proteins were coupled with a synthetic peptide homologous to a nuclear-transport-defective T antigen. The largest protein-peptide conjugate efficiently transported was ferritin (M, 465,000). The rate of transport was influenced by the number of peptides per molecule of carrier protein and, to some degree, by the size of the carrier protein. Transport of some conjugates was almost complete in 15 min at room temperature.