Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals.

Abstract

The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus EIA, and Saccharomyces cerevisiae MATh2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, plOO, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.Specific transport of proteins into the nucleus is an important requirement for the integrity of that organelle. Specificity requires the presence of both a nuclear transport signal and the cellular proteins that recognize that signal. Many nuclear transport signals have been defined by mutation and then by their ability as synthetic peptides (15,22) or as gene fusions (4,9,16,20,25,27,28,33,35,37,40) to direct nuclear localization of nonnuclear proteins. Unlike transport into the secretory pathway, nuclear transport allows movement without proteolytic removal of the signal, which permits repetitive transport of the protein. These signals are characteristically short amino acid sequences with a high number of basic residues. Variations in the number, the sequence, and the position of the nuclear transport signals have been shown to affect the rate of nuclear uptake (5, 10,22,23,36). There is no consensus nuclear transport signal, which is also the case for transport into the endoplasmic reticulum. Despite the lack of homology and apparent flexibility between nuclear transport signals, decreased transport efficiency occurs when key amino acids are varied (19,21,23). This combination of heterogeneity and stringency in nuclear transport signal sequences raises the question of what receptor flexibility and heterogeneity are needed for signal recognition.Nuclear transport occurs at nuclear pores, which create aqueous, octagonal channels in the double-membraned nuclear envelope (39). Pores restrict the movement of large proteins yet allow free diffusion of small proteins and macromolecules. Electron microscopic st...

Ad-Amino Acid Peptide Inhibitor of NF-κB Nuclear Localization Is Efficacious in Models of Inflammatory Disease

“…In addition, we have also synthesized another peptide, BMS 205820, that contains the NLS from SV40 large T Ag (16,40). To evaluate the effect of the peptides on NF-B nuclear translocation, we performed EMSA using nuclear extracts from 70Z/3 cells.…”

A hydrophobic protein sequence can override a nuclear localization signal independently of protein context.

“…While it was originally assumed that smaller nuclear proteins (<20 to 30 kDa) (14) would behave like small inert macromolecules and diffuse passively through the aqueous channel of the nuclear pore complex, a recent report by Breeuwar and Goldfarb (6) demonstrated that smaller nuclear proteins also use a receptor-facilitated import pathway. NLS-binding proteins have been detected by various assays in both nuclear and cytoplasmic compartments (1,33,54,59; for reviews, see references 17 and 52), and at least one cytoplasmic proteinaceous factor has been shown recently to be required for binding of nuclear proteins to the nuclear envelope in a cell-free assay (38) …”

Targeting of a cytosolic protein to the nuclear periphery.

“…It was shown that nuclear proteins can bind to components located at the nuclear periphery and the nuclear pores (Newmeyer and Forbes, 1988;Akey and Goldfarb, 1989) and these nuclear envelope constituents may be receptors of the nuclear localization signals. In addition, cytosolic factors, which also specifically bind to nuclear proteins, have been identified and a role in nuclear transport has been suggested (Adam et al, 1989;Yamasaki et al, 1989;Newmeyer and Forbes, 1990). However, the mechanism by which components of the nuclear pore complexes or putative receptors for nuclear proteins associated with the nuclear pores reach their correct subnuclear location, is still unknown.…”