ROLE OF THE NUCLEAR ENVELOPE IN INTRACELLULAR PROTEIN SORTING

核转运在调节其水平和活动中起着关键作用

Nuclear transport plays a key role in regulating the levels and activities of transcription factors, nuclear kinases, steroid hormone receptors and DNA replication factors. The nuclear pore complex (NPC) mediates the transport of mRNA and protein across the nuclear envelope. Previously, we described a family of phosphorylated glycoproteins which are components of the NPC. Interestingly, changes in the degree of phosphorylation and glycosylation occur during the cell cycle that may be associated with assembly and disassembly of nuclear pores. The major nuclear pore protein, p62 has been studied in greater detail. In addition, the enzyme which mediates O-linked GlcNAc addition to p62 has been purified and microsequenced. A homolog of the O-linked GlcNAC transferase has been identified in caenorhabditis elegans which may allow genetic manipulation of the enzyme in an intact organism. Using digitonin permeabilized cultured cells, we have studied nuclear and nucleolar transport of fluorescent conjugates bearing the appropriate localization signal. Such conjugates concentrated in the nucleus and nucleolus, respectively, when supplied with a cytosolic fraction and ATP. Nuclear Transport requires both Ca+2 and GTP suggesting that nuclear transport may be coordinately regulated with other signal transduction pathways during cellular activation. We have extended these studies using patch-clamp electrophysiology to examine regulated ion flow through the NE of cardiac myocytes. We detected large conductance ion channel activity at the NE resembling that of gap junction channels. Activation of these channels required cytosolic factors. The channels were blocked by GTP- gamma-S, wheat germ agglutinin and nuclear pore-specific antibodies. Thus, the large conductance nuclear ion channel activity is likely to be intrinsic to nuclear pores. Using recombinant TATA-binding protein, c-Jun, NF-kappaB and SP1 it was possible to demonstrate transcription factor translocation through the nuclear pore channel by patch-clamp measurements and atomic force microscopy. These observations strong support the notion of a nuclear pore channel that can be measured using elecrophysiological means. Since these measurements can be made in real-time it should be possible to examine conformational alterations in the nuclear pore during transport.