The novel ATP-binding cassette protein ARB1 is a shuttling factor that stimulates 40S and 60S ribosome biogenesis

Abstract

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.

FIG. 1.

Depletion of ARB1 reduces abundance of 40S ribosomal subunits. (A) The P_GAL-UBI-R-FH-ARB1 strain YDH226 was grown in SC_Gal-Leu medium to an A₆₀₀ of ∼1.2 (lane 1), and a portion of the culture was shifted to SC_Glu-Leu medium for 1 h (lane 2) or 2 h (lane 3). Twenty micrograms of WCEs (lanes 1 to 3) and 0.1 μg of pure FH-ARB1 (lane 4) were subjected to Western analysis with antibodies against the His₆ epitope. (B) Serial dilutions of YDH226 and WT strain BY4741 were spotted on yeast extract-peptone containing galactose (YPGal) and yeast extract-peptone containing glucose (YPGlu). (C to F) Strains BY4741 (C) and YDH226 (D to F) were grown in SC_Gal medium to an A₆₀₀ of ∼1.2 and shifted to SC_Glu medium for the times indicated in each panel. Cycloheximide was added at 50 μg/ml 5 min before harvesting, and WCEs were prepared in the presence of 10 mM Mg⁺² and resolved by velocity sedimentation through 7 to 47% sucrose gradients. Positions of 40S, 60S, 80S, and polysomes are indicated on the A₂₅₄ tracings. (G and H) BY4741 (WT) and YDH226 were grown in SC_Glu-Leu for 18 h as described for panels C to F except that cycloheximide was omitted and WCEs were prepared in the absence of Mg⁺² and resolved by velocity sedimentation through 15 to 30% sucrose gradients. The mean ratios of total 40S/60S subunits determined from replicate experiments are indicated with their standard errors.

FIG. 2.

Depletion of ARB1 leads to defects in 35S pre-rRNA processing and a deficit of 18S rRNA. (A to F) Strains BY4741 (WT) and YDH226 (P_GAL-UBI-R-FH-ARB1) were cultured as described in the legend of Fig. 1 for the indicated times in SC_Glu (Glu) medium, and total RNA was extracted and subjected to Northern analysis using the probes indicated along the side of each blot. (G) Schematic diagram of the pre-rRNA processing pathway. The 35S pre-rRNA contains the sequences for mature 18S, 5.8S, and 25S rRNAs, separated by the two internal transcribed spaces (ITS) and flanked by the external transcribed spaces (ETS). The processing sites are indicated by uppercase letters A to E. The annealing positions of probes 1 to 6 are depicted beneath all of the rRNA species which they detect.

FIG. 3.

Pulse-chase analysis further shows defective pre-rRNA processing in ARB1 depletion cells. Strains YDH209 (WT) and YDH226 (P_GAL-UBI-R-FH-ARB1) were grown in SC_Gal-Met medium to an A₆₀₀ of 1.2, then shifted to SC_Glu-Met medium for 18 h. Cells were labeled with [methyl-³H]methionine for 2 min and subsequently chased with 4 ml of 1 mg/ml unlabeled methionine for the time points indicated. Total RNA was isolated, and samples containing 70,000 dpm were separated on a 1.2% agarose-formaldehyde gel, transferred to a nylon membrane, and exposed to film. Lanes 9 and 10 depict a longer exposure of lanes 5 and 6.

FIG. 4.

Evidence that ARB1-GFP shuttles from nucleus to cytoplasm and is required for nuclear export of pre-40S ribosomes. (A) The LMB-sensitive strain crm1-T539C containing chromosomal ARB1-GFP (YDH338) (rows 1 and 2) was grown in YPD to an A₆₀₀ of ∼0.5. The crm1-T539C strain carrying plasmid-borne RPL25-GFP (YDH332) (rows 3 and 4) and a CRM1 strain carrying NOP1-GFP on a low-copy-number plasmid (YDH315) (row 5) were grown in SC_Glu-Ura to an A₆₀₀ of 1.0 and then diluted in YPD at an A₆₀₀ of 0.1 and grown to an A₆₀₀ of ∼0.5. The cells were incubated for 15 min in the presence or absence of LMB, fixed, and incubated with DAPI (4′,6′-diamidino-2-phenylindole) to stain the nuclear DNA. The GFP fusion proteins were visualized by fluorescence microscopy of living cells. Nomarski, phase-contrast imaging of cells. (B) The P_GAL-UBI-R-FH-ARB1 strain YDH226 carrying plasmid-borne RPS2-GFP was grown in SC_Gal-Ura medium to an A₆₀₀ of ∼1.2, and half of the culture was shifted to SC_Glu-Leu medium for ∼18 h. Fluorescence microscopy was conducted as described for panel A. Arrows indicate the fluorescent foci in the nuclei. (C) Enlarged view by confocal laser microscopy of the cells cultured in SC_Glu-Leu medium and described in panel B.

FIG. 5.

ARB1 cosediments with 40S, 60S, and 80S or 90S ribosomal species. Strains F1205 (ARX1-TAP) (B), F1207 (RIO2-TAP) (A), and YDH1014 (IMP4-myc₁₃) (C) were grown at 30°C to an A₆₀₀ of ∼1.2 in YPD medium and treated with cycloheximide (50 μg/ml) for 5 min before harvesting. WCEs were prepared in buffer containing 1.5 mM Mg⁺² and resolved by velocity sedimentation through 7 to 47% sucrose gradients in buffer containing 1.5 mM Mg⁺². The gradients were collected with continuous scanning at 254 nm, and fractions were subjected to Western analysis using antibodies against protein A for the TAP-tagged proteins (A and B), myc₁₃ for the myc₁₃ tagged IMP4 protein (C), or the other proteins indicated on the left. Lanes 1 to 15, gradient fractions from top to bottom; lane 16 in panels A and B and lane 17 in panel C contain 100 ng of purified FH-ARB1 protein. Lane 16 in panel C is blank.

FIG. 6.

ARB1 is physically associated with 40S and 60S processing factors. WT BY4741 or its derivatives containing chromosomal DED1-myc₁₃, ZUO1-myc₁₃, CBF5-myc₁₃, or TIF6-myc₁₃ (A) or LSG1-myc₁₃, SCP160-myc₁₃, or ARX1-myc₁₃ (B) were grown to an A₆₀₀ of ∼1.5 in YPD medium. WCEs were immunoprecipitated with anti-myc antibodies, and the immune complexes were subjected to Western analysis using antibodies against ARB1, myc epitope, GCD11, or 60S subunit protein RPL39, as indicated on the right. I, 1/100 of the input WCE extract; P, pellet fraction. The molecular sizes in kilodaltons are indicated on the left.

FIG. 7.

Signature sequences in the ABCs of ARB1 are essential in vivo. (A) The primary structure of ARB1 is depicted schematically with amino acid positions and the locations of mutations made in conserved residues (in italics) shown at the top, regions of sequence similarity to ATP-binding domains in other ABC proteins are shaded, and locations of the Walker A and B motifs and signature sequences (S) are indicated with black (ABC1) or white (ABC2) rectangles. The predicted interactions that would stabilize formation of a dimer between ABC1 and ABC2, sandwiching two molecules of ATP, are indicted with arrows. (B) Patches of an arb1Δ strain containing ARB1 URA3 plasmid pDH22 and LEU2 plasmids YCplac111 (empty vector; row 1), pDH144 (containing FH-ARB1-G229D-G230E-G519D; row 2), or pDH129 (containing FH-ARB1; row 3), were replica plated to SC_Glu-Leu-Ura (left panel) and medium containing 5-FOA (right panel). (C) WCEs of the strains described in panel B were grown in SC_Glu-Leu-Ura to an A₆₀₀ of ∼1.2, and 10 or 20 μg of WCE from cells containing pDH129 (lanes 1 and 2), pDH144 (lanes 4 and 5), or empty vector (lane 3) in addition to pDH22 were subjected to Western analysis with anti-His₆ antibodies. mt, mutant.