The Acidic C-terminal Domain of rna1p Is Required for the Binding of Ran·GTP and for RanGAP Activity

Abstract

The small GTP binding protein Ran is an essential component of the nuclear protein import machinery whose GTPase cycle is regulated by the nuclear guanosine nucleotide exchange factor RCC1 and by the cytosolic GTPase activating protein RanGAP. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae the RanGAP activity is encoded by the RNA1 genes which are essential for cell viability and nucleocytoplasmic transport in vivo. Although of limited sequence identity the two yeast proteins show a conserved structural organization characterized by an N-terminal domain of eight leucine-rich repeats, motifs implicated in protein-protein interactions, and a C-terminal domain rich in acidic amino acid residues. By analyzing the RanGAP activity of a series of recombinantly expressed rna1p mutant derivatives, we show that the highly acidic sequence in the C-terminal domain of both yeast proteins is indispensable for activating Ran-mediated GTP hydrolysis. Chemical cross-linking reveals that the same sequence in rna1p is required for rna1p⅐Ran complex formation indicating that the loss of GAP activity in the C-terminally truncated rna1p mutants results from an impaired interaction with Ran. The predominant species stabilized through the covalent cross-link is a rna1p⅐Ran heterodimer whose formation requires the GTP-bound conformation of Ran. As the acidic C-terminal domain of rna1p is required for establishing the interaction with Ran, the leucine-rich repeats domain in rna1p is potentially available for additional protein interactions perhaps required for directing a fraction of rna1p to the nuclear pore.The import of karyophilic proteins into the nucleus which occurs through the nuclear pore complex (NPC) 1 is an energydependent process specified by nuclear localization signals (NLSs) on the import substrate (for reviews see Refs. 1-4). Using digitonin-permeabilized HeLa cells as an in vitro transport system, it was shown that the import of substrate proteins is a multistep process depending on four cytosolic factors (5, 6) (for reviews see Refs. 7-10). Two of these factors form a heterodimeric complex serving as the NLS receptor. The smaller 60-kDa subunit of this complex, also known as importin ␣, is responsible for NLS binding, whereas the larger 97-kDa subunit, importin ␤, most likely mediates a targeting of the substrate-NLS receptor complex to the NPC. After docking the entire substrate-receptor complex is thought to travel through the NPC and then to dissociate close to or at the nucleoplasmic side of the pore (for review see Ref. 11). The two other factors identified in the in vitro transport system as essential components of the nuclear import machinery are the GTP-binding protein Ran/TC4 (herein referred to as Ran (12, 13)) and a small protein of 10 kDa, p10/NTF2. The latter can interact with NPC proteins, Ran, and importin ␤ and appears to be involved in the formation of a pentameric complex including p10, Ran in its GDP-bound form, the two NLS receptor subunits and a nuclear pore protein (14, 15).R...

Biochemical Characterization of the Ran-RanBP1-RanGAP System: Are RanBP Proteins and the Acidic Tail of RanGAP Required for the Ran-RanGAP GTPase Reaction?

“…In mammalian RanGAPs, it consists almost entirely of glutamate and aspartate. Haberland et al (26), using C-terminal deletion mutants of Schizosaccharomyces pombe and Saccharomyces cerevisiae RanGAP and phosphate release assays, suggested that the C terminus is required for GAP activity and Ran binding. It should be noted that the structure of RanGAP was not available at that time (30) and that those deletion constructs were based on incorrectly assigned LRRs which in turn were based on the structure of RNase A inhibitor (37).…”

“…Furthermore, while RanBDs were shown to costimulate RanGAP-catalyzed hydrolysis (10,72), the structure of the ternary complex and multiple turnover kinetics suggested that the catalytic machinery was entirely located on Ran. Previously, it was suggested that the conserved C-terminal highly acidic region of RanGAP is required for Ran binding and for stimulation of GTP hydrolysis (26). Although this acidic region was not visible in the structure, it did not appear to participate in catalysis.…”

Phosphorylation of RanGAP1 Stabilizes Its Interaction with Ran and RanBP1

“…The func-tional roles of the acidic domain of RanGAP1 continue to remain controversial, despite the fact that the domain is conserved over different phyla. The acidic domain of yeast rna1p was reported to be essential for Ran-GTP binding and/or RanGAP1 activity (Haberland et al, 1997, Traglia et al, 1989. A recent report, however, indicated that the acidic region of yeast Rna1p was dispensable for GAP activity and Ran binding, but instead, was essential for microtubule formation during mitosis (Seewald et al, 2003).…”

Scribble protein domain mapping reveals a multistep localization mechanism and domains necessary for establishing cortical polarity

“…Two good candidate effectors are Ran GTPase and Pins. LRRs are known to physically interact with Ran, which promotes spindle assembly through several target proteins (Dasso, 2001;Haberland et al, 1997;Haberland and Gerke, 1999). For example, Ran stimulates the activity of NuMA (a microtubule motor accessory protein that promotes spindle assembly) by destabilizing inhibitory complexes associated with NuMA (Dasso, 2001;Dasso, 2002;Nachury et al, 2001;Wiese et al, 2001).…”