Histone-poly(A) hybrid molecules as tools to block nuclear pores

Abstract

Histone-poly(A) hybrid molecules were used for transport experiments with resealed nuclear envelopes and after attachment of a cleavable cross-linker (SASD) to identify nuclear proteins. In contrast to histones, the hybrid molecules cannot be accumulated in resealed nuclear envelopes, and in contrast to poly(A), the export of hybrids from preloaded nuclear envelopes is completely impaired. The experiments strongly confirm the existence of poly(A) as an export signal in mRNA which counteracts the nuclear location signals (NLS) in histones. The contradicting transport signals in the hybrid molecules impair translocation through the nuclear pore complex. The failure to accumulate hybrid molecules into resealed nuclear envelopes results from the covalent attachment of polyadenylic acid to histones in a strict 1:1 molar ratio. This was demonstrated in control transport experiments where radiolabeled histones were simply mixed with nonlabeled poly(A) or radiolabeled poly(A) mixed with nonlabeled histones. In comparison, control uptake experiments with histones covalently linked to a single UMP-mononucleotide are strongly enhanced. Such controls exclude the conceivable possibility of a simple masking of the nuclear location signal in the histones by the covalent attached poly(A) moiety. Photoreactive histone-poly(A) hybrid analogs serve to identify nuclear envelope proteins--presumably in the nuclear pore--with molecular weights of 110, 80, and 71.4 kDa.

Nuclear transport of histone 2b in mammalian cells is signal- and energy-dependent and different from the importin α/β-mediated process

“…Histones were isolated from calf thymus following the method of Johns (1967) and histone fractions were further purified, as previously described, using reversed-phase HPLC (Hill and Thomas 1990;Cremer et al 1995). Purified H2b and H2a were dialyzed against 20 mM Na 2 HPO 4 , pH 7.5, and adjusted to 1 mg protein/ml.…”