The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

Abstract

Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP(6))-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP(6) stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP(6) regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

Figure 1.

Nup159 promotes Dbp5 release of ADP. (A) Equilibrium binding assays show an increase in initial rate of Dbp5–¹⁴C-ADP binding: Dbp5 (1 μM) was incubated with increasing concentrations of ¹⁴C-ADP (0.5–150 μM) over the course of 24 h. Samples were removed at several time points and used in filter-binding assays. Background integrated density signals of ¹⁴C-ADP at each concentration were subtracted from the Dbp5-bound ADP signal. As listed in the table, initial velocities of binding (fraction ¹⁴C-ADP bound per minute) were calculated during the linear phase of the binding curve at each Dbp5:¹⁴C-ADP ratio tested. (B) Dbp5 does not release bound ADP after equilibration: After 24-h equilibration of 1 μM Dbp5 and 5 μM ¹⁴C-ADP:MgCl₂, excess unlabeled ATP:MgCl₂ or ADP:MgCl₂ (10 mM of each) was added, and filter-binding assays were conducted after an additional 24 h incubation. (C) Nup159-NTD does not promote release of the nonhydrolyzable ATP analog ADP-BeFx: After 26-h equilibration of 1 μM Dbp5 with 5 μM ¹⁴C-ADP-BeFx, either 1 μM Nup159NTD, 500 nM Gle1 with 200 nM IP₆, or 2 μM poly(A)₂₅ RNA was added, and filter-binding assays were performed at 24 h after addition. (D) Nup159-NTD specifically promotes Dbp5 release of bound ADP: After 24-h equilibration of 1 μM Dbp5 with 5 μM ¹⁴C-ADP:MgCl₂, either 1 μM Nup159-NTD, 500 nM Gle1 with 200 nM IP₆, or 2 μM RNA was added, and filter-binding assays were performed at 10 min and 24 h after addition. Two altered nup159 proteins (nup159^DD and nup159^VI) that do not bind Dbp5 in vitro were used as negative controls. (E) Increasing concentration of Nup159 promotes faster Dbp5 release of bound ¹⁴C-ADP. Dbp5 (1 μM) was incubated with 5 μM ¹⁴C-ADP for 24 h. Increasing concentrations of Nup159 were added and filter-binding assays were performed over the course of 2 h. All data points and bars represent at least three independent experiments with standard error bars.

Figure 2.

Gle1-IP₆ enhances Dbp5 binding to ATP. (A,B) Adenine nucleotide binding of Dbp5 versus dbp5^EQ: Filter-binding assays were conducted to assay for relative binding to ATP and ADP. Purified recombinant Dbp5 or dbp5^EQ (1 μM) was incubated with 1 μM ³²P-ATP:MgCl₂ or ¹⁴C-ADP:MgCl₂ and increasing concentrations of unlabeled nucleotide ranging from 0 nM to 5000 nM. Bars represent the averages of three independent experiments with standard error shown. (C) dbp5^EQ is inhibited for ATPase activity: Colorimetric ATPase-coupled enzyme assays were performed with purified recombinant 500 nM Dbp5 or dbp5^EQ, 1 mM ATP:MgCl₂, and 1 μM poly(A)₂₅ RNA. Gle1 (250 nM) was added with or without 100 nM IP₆. (D) Gle1-IP₆ promotes ATP loading of dbp5^E240Q: dbp5^E240Q (2 μM) was incubated with 10 nM α³²P-ATP in the presence of 2 μM Nup159-NTD, 1 μM Gle1 with 400 nM IP₆, or 4 μM poly(A)₂₅ RNA for 4 h. Filter-binding assays were performed. In all panels, bars represent the averages of three independent experiments with standard error bars.

Figure 3.

dbp5^RR mutant does not bind Nup159 and displays no in vivo growth defects. (A) Amino acid residue alignment for a portion of S. cerevisiae Dbp5 and Homo sapiens hDBP5. Black stars designate conserved arginine residues that were changed to aspartic acid residues (R256D and R259D) in Dbp5 to yield dbp5^RR. The open white star represents a conserved glutamate residue that was changed to glutamine (E240Q) to yield dbp5^EQ. Conserved DEAD-box family sequence motifs are boxed and identified below alignment. (B) Cytoplasmic mislocalization of GFP-dbp5^RR by live-cell microscopy: S. cerevisiae strains with DBP5 chromosomally deleted, expressing either GFP-DBP5 or GFP-dbp5^RR on plasmids, were used in live-cell fluorescent microscopy experiments to visualize subcellular localization of each protein. (Left) DIC image. (Right) GFP. Western blot analysis of yeast whole-cell extracts was conducted to test for relatively equal levels of expression of GFP-Dbp5 and GFP-dbp5^RR. Nup116 protein level was used as a loading control. (*) Degradation products of Nup116. (C) Nup159-NTD interaction in vitro with dbp5^RR is not detected: Soluble binding assays were conducted with 200 pmol of bacterially expressed recombinant His₆-Nup159-NTD and immobilized on Ni-NTA agarose resin and either recombinant Dbp5, dbp5^RR, or dbp5^EQ. Input and bound fractions were analyzed by SDS-PAGE and Coomassie staining. (*) Nup159-NTD degradation product. (D) Swapping the salt bridge interaction allows nup159^DD and dbp5^RR interaction: Soluble binding assays were conducted as in C with His₆-Nup159-NTD, His₆-nup159^VI-NTD, His₆-nup159^DD-NTD, Dbp5, or dbp5^RR. (E) dbp5^RR strain does not have growth defects: S. cerevisiae strains with DBP5 chromosomally deleted, expressing either wild-type DBP5 or dbp5^RR, were used to perform serial dilution growth assays at 23°C, 30°C, and 37°C.

Figure 4.

dbp5^RR does not require Nup159 for ADP release. (A) dbp5^RR has reduced binding affinity to ADP as compared with wild-type Dbp5: Dbp5 (1 μM) or dbp5^RR (1 μM) was incubated with 5 μM ¹⁴C-ADP and increasing concentrations of unlabeled ADP:MgCl₂. Filter-binding assays were performed to measure the relative fraction of ADP bound to each protein. Bars represent the averages of three independent experiments with standard error bars. (B) dbp5^RR does not require Nup159 for ADP release: α³²P-ATP was incubated with either Dbp5 or dbp5^RR and 400 μM poly(A)₂₅ RNA for 24 h to allow complete hydrolysis to α³²P-ADP to occur. After 24 h, samples for filter-binding assays were removed and the remaining reaction was stopped with proteinase K digestion. (Left) The digested samples were spotted onto thin-layer chromatography plates to monitor ATP hydrolysis. (Right) The portion of the samples removed for filter-binding assays were used to monitor the relative amount of ADP still bound to either Dbp5 or dbp5^RR. Bars represent the averages of three independent experiments with standard error shown. (C) dbp5^RR is active for ATP hydrolysis and Gle1-IP₆ stimulation: Colorimetric ATPase-coupled enzyme assays were performed as described in Figure 1B with 500 nM Dbp5 or 500 nM dbp5^RR. Gle1 (250 nM) with 100 nM IP₆ was added to measure relative levels of stimulation. The ATPase activity of each sample was normalized to the maximally stimulated Dbp5 with Gle1-IP₆ sample. Bars represent the averages of three independent experiments with standard error shown. (D) The dbp5^RR mutant complements the rat7ΔN temperature sensitivity. Yeast strains were grown at the permissive temperature and serially diluted on plates for growth at 23°C and 37°C. (E) The dbp5^RR nup42Δ double mutant has a synthetic fitness growth defect. Yeast strains were grown at the permissive temperature and serially diluted on plate for growth at 23°C and 37°C. (F) Nuclear poly(A) RNA accumulates in arrested dbp5^RR nup42Δ cells. In situ hybridization with an oligo(dT) probe for poly(A) RNA localization was conducted on dbp5^RR nup42Δ cells grown at 23°C and shifted to growth for 1 h at 37°C. DAPI staining marks the nucleus (left) and fluorescent signal for the poly(A) RNA (right).

Figure 5.

Dbp5 remodeling mechanism is mediated by a conformational change. (A) In vitro remodeling assay demonstrates that dbp5^EQ remodels similarly to wild-type Dbp5: In vitro constituted Nab2-RNPs were assembled as described in Tran et al. (2007) with 5′-end labeled ³²P-poly(A)₂₅. Nab2 (250 nM) was incubated with 10 nM RNA and 500 nM either wild-type Dbp5 or dbp5^EQ in the presence of 1 mM ATP:MgCl₂ or 1 mM ADP:MgCl₂. Electrophoretic mobility shift assays (EMSAs) were performed and products were visualized by autoradiography. (B) Quantification of Nab2-RNPs remodeling: Increasing concentrations of Dbp5 and dbp5^EQ were added to in vitro constituted Nab2-RNPs in the presence of ADP:MgCl₂ and filter-binding assays were performed. Densitometry was used to quantify the fraction of Nab2-RNPs that were remodeled at each concentration of protein. Bars represent the averages of three independent experiments with standard error bars. (C) dbp5^RR remodels in vitro constituted Nab2-RNPs less efficiently than wild-type Dbp5: EMSAs were performed as described above using 750 nM wild-type Dbp5, dbp5^E240Q, or dbp5^RR in the presence of 1 mM ADP:MgCl₂. The gel was exposed to autoradiography to detect the relative Nab2-RNP and free RNA. (D) Circular dichroism studies demonstrate nucleotide-dependent conformational changes: Near-UV spectral scans of Dbp5 with 1.5 mM ATP:MgCl₂, ADP:MgCl₂, or MgCl₂ alone were collected from 250-nm to 350-nm wavelengths. Spectra of buffer with 1.5 mM ATP:MgCl₂, ADP:MgCl₂, or MgCl₂ were subtracted from the Dbp5 spectra to remove interfering signals from the adenosine moiety. Θ represents molar elipticity, which is calculated as (100 × signal)/(pathlength × amino acid residues × mM concentration).

Figure 6.

Proposed mechanism of Dbp5 nucleotide cycle. The left panel illustrates in vivo steps, and nucleotide binding/hydrolysis are controlled by multiple binding partners. (Step 1) Bound to ATP and an mRNP, Dbp5 is primed for remodeling. Hydrolysis is stimulated by Gle1-IP₆, resulting in release of Pi and the remodeled mRNP, and generation of a conformationally changed ADP-bound Dbp5. (Step 2) Bound to ADP, Nup159 promotes ADP release, which confers another conformational change and places Dbp5 in an APO state. (Step 3) Gle1-IP₆ promotes the loading of ATP to allow another round of nucleotide cycling and remodeling. (Step 4) Bound to ATP, Dbp5 has the highest affinity for RNA. RNA binding and ATP hydrolysis release Gle1-IP₆. The right panel proposes a model for in vitro mimicking via ADP binding to the APO form of Dbp5, allowing mRNP remodeling in the absence of Gle1-IP₆. Once bound to ADP in vitro, Nup159 promotes nucleotide release.