HIV-1 NUCLEAR IMPORT: IN SEARCH OF A LEADER

Michael I. Bukrinsky¹ and Omar K. Haffar²

1The Picower Institute for Medical Research, Manhasset, NY 11030, ²Cytokine Networks Inc., 101 Elliott Ave West, Suite 428, Seattle, WA 98119

Received 11/7/97 Accepted 11/14/97

3. CELLULAR NUCLEAR IMPORT MACHINERY

Nuclear transport of macromolecules occurs through the nuclear pore complexes and is controlled by the nuclear localization signals (NLSs). The most common type of NLSs is a short stretch of basic amino acids that introduce an overall net positive charge crucial for nuclear targeting properties of these sequences (reviewed in (16)). Import of NLS-containing proteins across the nuclear pore complex is mediated by karyopherin alphabeta heterodimers (also termed NLS receptor/importin) which bind NLS-containing proteins in the cytosol and target them to the nucleus (17-23). Karyopherin alpha binds the NLS (17) whereas karyopherin beta enhances the affinity of alpha for the NLS (24) and mediates docking of karyopherin-NLS protein complexes to nucleoporins (a collective term for nuclear pore complex proteins) that contain FG peptide repeats (20,24,25). A different import pathway is mediated by the M9 domain, which is the import signal for hnRNP such as A1, and employs the transportin- (a homologue of karyopherin beta), rather than karyopherin alphabeta-mediated, pathway (26-29).

The small GTP binding protein, Ran (30,31), is a key regulator of the import process. Ran switches between the GDP- and GTP-bound states by nucleotide exchange and GTP hydrolysis. Because the intrinsic rates of these reactions are very low, the nucleotide associated with Ran, and hence the state of Ran's activation, is determined by the presence of RCC1 (Ran's major nucleotide exchange factor (32)) and RanGAP1 (the only known RanGTPase-activating protein (33,34)). RCC1 is chromatin-bound (35), while RanGAP1 is excluded from the nucleus (36). Therefore, the concentration of Ran-GTP is high in the nucleus and low in the cytoplasm. It is believed that this gradient is used to provide direction to the nucleo-cytoplasmic exchange. In particular, by directly binding to karyopherin beta in the nucleoplasm, Ran-GTP disassembles the import complex (24) and thus terminates the import process (37). It also stimulates assembly of the karyopherin alpha complex with CAS, a recently discovered export factor (38), thus promoting re-export of karyopherin alpha into the cytoplasm for recycling. A family of Ran-binding proteins facilitates the function of Ran. These include RanBP1, which activates RanGTPase (39,40) and also stabilizes the interaction of Ran-GDP with karyopherin betaduring translocation through the pore (41), and RanBP2 (Nup358), which may be the initial docking site for nuclear protein import (42,43).

In addition to karyopherins and Ran, several other soluble proteins are involved in nuclear import, although their mechanism of action is less defined. The nuclear import factor p10 (also termed NTF2) (44,45) appears to coordinate the activity of Ran by binding Ran-GDP into a complex with nucleoporin-docked karyopherins (46). Heat-shock protein 70 (Hsp70, Hsc70), as well as some as yet uncharacterized cytoplasmic factors, may act to facilitate the interaction between the NLS and karyopherin alpha (47,48). The ectopic expression of human Hsp70 in mouse cells complemented the defective import of a mutant SV40 large T antigen (49), and the depletion of Hsp70 from cytosolic extracts prevented import (50,51).