Nuclear Envelope Vesicles as an In Vitro Model System to Study Nucleocytoplasmic Transport of Proteins and Ribonucleic Acids

Abstract

We describe a procedure for the preparation of nuclear envelope vesicles from rat liver nuclei. These vesicles contain the components of the nuclear envelope, e.g. the pore-complex-lamina fraction, and a residual DNA content of 1.5%. Typical preparations consist of about 90% vesicles with the vesicular character of these envelopes shown by means of microscopic and biochemical studies. Uptake measurements using nuclear and non-nuclear proteins show that the vesicles have a high affinity only for proteins that are specific for the nuclear compartment and indicate that the nuclear envelope affects the uptake kinetics and increases the capacity for nuclear proteins. The nuclear envelope vesicles contain the translocation mechanism for mRNA and show an unidirectional transport of mRNA from the vesicular interior into the medium; this efflux can be stimulated by ATP. Because the vesicles are virtually free of the components of the nuclear interior, but retain properties of intact nuclei, we believe that they are a valuable model system to investigate the structures and mechanisms involved in the regulation of nucleocytoplasmic transport processes.