It has been estimated that more than a million macromolecules per minute are transported between the nucleus and the cytoplasm in an active eukaryotic cell. These macromolecules include histones, ribosomal proteins and ribosomal subunits, transcription factors, and mRNA molecules. The transport is bidirectional and occurs through the nuclear pore complexes (NPCs). These are complex structures with a mass approximately 15 times that of a ribosome and are composed of aggregates of about 30 different proteins. The minimal diameter of an NPC is approximately 9 nm. Molecules smaller than about 40 kDa can pass through the channel of the NPC by diffusion, but special translocation mechanisms exist for larger molecules.
Here we shall mainly describe current knowledge about the nuclear import of certain macromolecules. The general picture that has emerged is that proteins to be imported (cargo molecules) carry a nuclear localization signal (NLS). One example of an NLS is the amino acid sequence (Pro)2-(Lys)3 Arg-Lys-Val, which is markedly rich in basic residues. A cargo molecule containing an NLS interacts with importin, a soluble protein which has two subunits, termed α and β. For the transport of proteins into the nucleus, importin usually functions as a heterodimer, importin α/β. The α sub unit binds to the NLS and the complex docks transiently at the NPC via interaction with the β subunit. Another family of proteins called Ran plays a critical regulatory role in the inter action of the complex with the NPC and in its translocation through the NPC. Ran proteins are small monomeric nuclear GTPasesand, like other GTPases, exist in either GTP-bound or GDP-bound states. They are themselves regulated by guanine nucleotide exchange factors (GEFs), which are located in the nucleus, and Ran GTPase-accelerating proteins (GAPs), which are predominantly cytoplasmic. The GTP-bound state of Ran is favored in the nucleus and the GDP-bound state in the cytoplasm. The conformations and activities of Ran molecules vary depending on whether GTP or GDP is bound to them (the GTP-bound state is active; see discussion of G-proteins in Chapter 42). The asymmetry between nucleus and cytoplasm—with respect to which of these two nucleotides is bound to Ran molecules—is thought to be crucial in understanding the roles of Ran in transferring complexes unidirectionally across the NPC. When cargo molecules are released inside the nucleus, the importins recirculate to the cytoplasmto be used again. Figure 1 summarizes some of the principal features in the above process.
Fig1. The entry of a protein into the nucleoplasm.A cargo molecule (C) in the cytoplasm interacts via its nuclear localization signal (NLS) to form a complex with importin (I). This complex binds to Ran (R)·GDP and traverses the nuclear pore complex (NPC) into the nucleoplasm. In the nucleoplasm, Ran·GDP is converted to Ran·GTP by guanine nuclear exchange factor (GEF), causing a conformational change in Ran which releases the cargo molecule. The I-Ran·GTP complex then leaves the nucleoplasm via the NPC to return to the cytoplasm. Here I is released by the action of GTPase-accelerating protein (GAP), which converts GTP to GDP, enabling it to bind to another C. The Ran·GTP is the active form of the complex, with the Ran·GDP form is inactive. Directionality is believed to be conferred on the overall process by the dis sociation of Ran·GTP in the cytoplasm.
Proteins similar to importins, referred to as exportins, are involved in the export of many macromolecules (various proteins, tRNA molecules, ribosomal subunits, and certain mRNA molecules) from the nucleus. Cargo molecules for export carry nuclear export signals (NESs). Ran proteins are involved in this process also, and it is now established that the processes of import and export share a number of common features. The family of importins and exportins are referred to as karyopherins.
Another system is involved in the translocation of the majority of mRNA molecules. These are exported from the nucleus to the cytoplasm as messenger ribonucleoprotein (mRNP) complexes attached to a protein termed mRNP exporter that carries mRNP molecules through the NPC. Ran is not involved. This system appears to use the hydrolysis of ATPby an RNA helicase (Dbp5) to drive translocation.
Other small monomeric GTPases (eg, ARF, Rab, Ras, and Rho) are important in various cellular processes such as vesicle formation and transport (ARF and Rab; see later), certain growth and differentiation processes (Ras), and formation of the actin cytoskeleton (Rho). A process involving GTP and GDP is also crucial in the transport of proteins across the membrane of the ER.
