Antigen Nuclear Localization Signal Research Articles

Nuclear protein import: Specificity for transport across the nuclear pore

Transport of proteins into the cell nucleus is thought to require specific localization sewuences 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; M r 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, 9887–98941 and in vivo. Microinjection of WGA into the cytoplasm of living cells did not alter the diffusion of dextran ( M r 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.

Organ development in the collagen I — Deficient Mov-13 mouse: [formula omitted] ∗ Inst. f. Molekularbiologie, Salzburg, Austria § Max-Planck-Inst. f. Biochemie, München; + Heinrich-Pette-Inst. Hamburg, Germany

Moloney murine leukemia virus (M-MuLV) proviruses carrying integrase (IN) protein tagged either with a simian virus 40 (SV40) nuclear localization signal (NLS) or various antigenic epitopes were generated. Hexahistidine (His6), hemagluttinin (HA), or two consecutive HA sequences (2XHA) were fused to the C-terminus of IN as antigenic markers. These epitope-tagged IN proteins were stably expressed through multiple rounds of infection. The IN-His6, IN-HA, and IN-2XHA proteins, purified from virus, could be immunoprecipitated with antibodies against His6 and HA, respectively. An M-MuLV provirus encoding the SV40 large T antigen NLS fused to IN at the same position as the epitope tags was also passaged through cells. In contrast to the stability of the epitope tags, the SV40 NLS sequence was rapidly mutated by a frameshift mutation that introduced negatively charged amino acids into the basic NLS. The instability of the NLS suggests that the strong nuclear localization of the IN-SV40 NLS may have detrimental effects on virus assembly. These observations have implications for studying nuclear transport properties of M-MuLV and for engineering a murine-based retroviral vector for gene therapy.