Ultrastructural nuclear import assay.
Electron microscopy (EM) has been used for several decades to study the mechanisms of nuclear transport. In early studies of nuclear import, gold-conjugated nuclear proteins were microinjected into cells and followed by EM. As the components of the nuclear pore complex (NPC) and soluble mediators of nuclear import were cloned and characterized, gold-conjugated antibodies were utilized to sublocalize the components of the nuclear transport machinery by immuno-EM. Further, gold-conjugated recombinant proteins were used to probe permeabilized cells or isolated nuclear envelopes and characterize binding sites for these proteins at the NPC. More recently, recombinant gold-conjugated nuclear proteins were used in in vitro nuclear import assays to help dissect the mechanisms of nuclear import. We have used this ultrastructural nuclear import assay to study the nuclear import of the transcription factor PU.1. The results showed that this import requires energy but is carrier-independent. In the presence of energy, gold-conjugated PU.1 shifted to the nuclear side of the NPC and the inside of the nucleus. In conjunction with biochemical assays, these results indicated that this shift involved Ran-dependent binding of PU.1 to NUP153, a nucleoporin situated at the nuclear side of the NPC. Here we describe in detail the methods used in the ultrastructural nuclear import assay including preparation of recombinant protein, gold conjugation, in vitro nuclear import assay, electron microscopy, and data analysis.
Zhong
Hualin
H
Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10021, USA.
Shio
Helen
H
Yaseen
Nabeel R
NR
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D001681:Biological Assay; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D009685:Nuclear Envelope / Q000378:metabolism / Q000648:ultrastructure; D022022:Nuclear Pore / Q000648:ultrastructure; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000378:metabolism* / Q000648:ultrastructure; D021381:Protein Transport / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000648:ultrastructure; D015534:Trans-Activators / Q000378:metabolism / Q000648:ultrastructure; D000099232:Proto-Oncogene Protein Spi-1
D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C079377:NUP153 protein, human