Nuclear protein import: specificity for transport across the nuclear pore

Abstract

Transport of proteins into the cell nucleus is thought to require specific localization sequences and may be mediated by nuclear pores. Following microinjection into fused cultured cells, nuclear protein import was directly monitored by fluorescence microscopy using B-phycoerythrin (PE; Mr 240,000) coupled to synthetic peptides corresponding to the simian virus 40 (SV-40) large T antigen nuclear localization signal. Peptides with a single amino acid replacement found in a cytoplasmic mutant of T antigen (cT) failed to promote uptake. Further studies with deletion peptides revealed the minimum sequence requirements for efficient nuclear import of PE conjugates to be similar to those previously defined genetically for large T antigen itself. No competitive inhibition of uptake was observed in cells expressing nuclear or cytoplasmic T antigen. Nuclear import was time- and temperature-dependent. The lectin wheat germ agglutinin (WGA) binds to glycoproteins bearing O-linked GlcNAc on the cytoplasmic face of the nuclear pore in vitro [J.A. Hanover et al. (1987) J. Biol. Chem. 262, 9887-9894] and in vivo. Microinjection of WGA into the cytoplasm of living cells did not alter the diffusion of dextran (Mr 10,000) into the nucleus, but blocked the uptake of PE conjugates. This inhibition was reversed when a competing saccharide was introduced into the cytoplasm.