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. 1989 Dec;86(23):9327–9331. doi: 10.1073/pnas.86.23.9327

Interaction of a nuclear location signal with isolated nuclear envelopes and identification of signal-binding proteins by photoaffinity labeling.

J O Benditt 1, C Meyer 1, H Fasold 1, F C Barnard 1, N Riedel 1
PMCID: PMC298488  PMID: 2556708

Abstract

The nuclear envelope (NE) separates the two major compartments of eukaryotic cells, the nucleus and the cytoplasm. Recent studies suggest that the uptake of nuclear proteins into the nucleus is initiated by binding of nuclear location signals (NLSs) contained within these proteins to receptors in the NE, followed by translocation through the nuclear pore complex. To examine the binding step without interference from intranuclear events, we have used a system consisting of (i) purified rat liver NEs fixed onto glass slides and (ii) the prototype simian virus 40 large T antigen (SV40 T) NLS conjugated to nonnuclear carrier proteins, and we have visualized the receptor-ligand interaction by indirect immunofluorescence. In this system, incubation of isolated NEs with the wild-type SV40 T NLS conjugate with carrier proteins resulted in binding that was signal sequence-dependent, could be competitively blocked with excess conjugated and unconjugated wild-type peptide, did not require ATP, and was not affected by the transport-inhibiting lectin wheat germ agglutinin. In contrast, only minimal binding was observed with a mutant SV40 T NLS conjugate. These results are consistent with those obtained in other, more complex in vitro systems and suggest that binding of the SV40 T NLS is receptor-mediated. Binding is largely abolished by extraction of the NE with the nonionic detergent Triton X-100, suggesting that the receptor is soluble in detergent. We find in the Triton X-100 supernatant four major NLS-binding proteins with apparent molecular masses of 76, 67, 59, and 58 kDa by photoaffinity labeling with a highly specific crosslinker, azido-NLS. The reduced complexity of the system described here should be useful for the functional study of other potential NLSs for the identification and isolation of their binding sites and for the screening of antibodies raised against these binding sites.

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