Volume 26, Issue 1, Pages (April 2007)

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Volume 26, Issue 1, Pages 51-62 (April 2007) Nuclear Export of Ribosomal 60S Subunits by the General mRNA Export Receptor Mex67-Mtr2  Wei Yao, Daniela Roser, Alwin Köhler, Bettina Bradatsch, Jochen Baßler, Ed Hurt  Molecular Cell  Volume 26, Issue 1, Pages 51-62 (April 2007) DOI: 10.1016/j.molcel.2007.02.018 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 In Vivo, the Mex67-Mtr2 Heterodimer Is Associated with Late Pre-60S Particles (A) Tandem affinity purification (TAP) of pre-60S particles via the indicated TAP-tagged bait proteins. Eluates were analyzed by SDS-PAGE and Coomassie staining (upper part) or western blotting (lower part) using antibodies against Mex67, Nmd3, and Rpl3. S, protein standard. Lanes 1–4, preribosomal bait proteins (●). Rpl, ribosomal L proteins. (B) Salt-sensitive binding of Mex67-Mtr2 to the pre-60S particle. TAP of Arx1 particle was performed in buffer with increasing concentrations of NaCl (lane 1–4). Eluates were analyzed by SDS-PAGE and Coomassie staining (upper part; indicated are the positions of Arx1, Rpl3, and Rpl4) or western blotting (lower part) using antibodies against Mex67, Mtr2, Nmd3, and Rpl3. Molecular Cell 2007 26, 51-62DOI: (10.1016/j.molcel.2007.02.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 The Yeast-Specific Loops of Mex67 and Mtr2 Are Involved in Binding to Pre-60S Particles (A) In vivo role of the long loops in the NTF2-like middle domain of Mex67 and Mtr2 for cell growth. Schematic drawing of the Mex67 and Mtr2 domain organization (upper panel). Mex67 consists of an N-terminal (N), leucine-rich repeat (LRR), NTF2-like middle, and UBA-like carboxy-terminal domain. Mtr2 consists only of an NTF2-like domain. Indicated in the Mex67 middle domain and Mtr2 are the long internal loops. Growth analysis of wild-type and mutant mex67 and mtr2 strains (lower panel). Serial dilutions of the indicated cells were spotted onto YPD plates and grown for 3 days at the indicated temperatures. (B) Mex67Δloop is strongly reduced in the TAP-purified Arx1particle. Arx1-TAP was affinity purified under standard TAP conditions (100 mM NaCl) from cells expressing wild-type Mex67 or mutant Mex67Δloop, Mex67 loop KR>AA, and Mex67ΔloopK343E as well as wild-type Mtr2 or mutants mtr2Δloop116–137 and mtr2 RR>DD. Eluates were analyzed by SDS-PAGE and Coomassie staining and western blotting using antibodies against Mex67, Mtr2, Nmd3, and Rpl3. A protein standard is shown on the left, and prominent Arx1, Rpl3, and Rpl4 are indicated on the right. (C) Residual binding of Mex67Δloop to pre-60S particles under lower salt conditions. Arx1-TAP was affinity purified under standard (100 mM NaCl) or lower salt conditions (50 mM NaCl) from cells expressing wild-type Mex67 or mutant Mex67Δloop. Eluates were analyzed by SDS-PAGE and western blotting using antibodies against Mex67 and Rpl3. Molecular Cell 2007 26, 51-62DOI: (10.1016/j.molcel.2007.02.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 5S rRNA Binds to the Loop-Confined Surface on the NTF2-like Mex67-Mtr2 Heterodimer (A) 5S rRNA binds to the NTF2-like domain of the Mex67-Mtr2 heterodimer. Full-length Mex67-Mtr2 complex and middle domain Mex67-Mtr2 (Mex67 MD-Mtr2) heterodimer were affinity purified and used in RNA-electrophoresis mobility shift assays using in vitro-transcribed yeast 5S rRNA (∼150 ng per lane) or total yeast tRNA (∼250 ng per lane). No protein (lane 1) and increasing amounts (50 ng–1 μg) of recombinant proteins (lanes 2–8). RNAs were visualized on a 6% polyacrylamide gel by ethidium bromide staining. Nonshifted RNA and formed RNA-protein complexes (band-shift 5S rRNA) are indicated. A faster-migrating band-shifted species was generated at low Mex67-Mtr2 concentrations. The slower-migrating band-shifted species formed at higher Mex67-Mtr2 concentrations may correspond to binding of Mex67-Mtr2 to a second region within 5S rRNA. The protein input was analyzed by SDS-PAGE and Coomassie staining. S, protein standard; 1, Mex67-Mtr2; 2, Mex67 middle domain (MD)-Mtr2. (B) Only intact 5S rRNA efficiently binds to Mex67-Mtr2. The 5S rRNA is 121 nt in length and contains helix and loop structures (3D structure is shown on the left). In vitro-transcribed full-length, 5′ half (1–69 nt) and 3′ half (67–111 nt) 5S rRNA were analyzed in the RNA-electrophoresis mobility shift assay using wild-type Mex67-Mtr2 (middle panel). Protein input was analyzed by SDS-PAGE and Coomassie staining (right panel). (C) The Mex67 loop is involved in mediating binding of the Mex67-Mtr2 heterodimer to 5S rRNA. For protein input (right panel), purified recombinant wild-type Mex67-Mtr2 (lanes 1), mutant complexes Mex67 loop KR>AA-Mtr2 (lane 2), Mex67-Mtr2 RR>DD (lane 3), Mex67Δloop-Mtr2 (lane 4), and Mex67ΔloopK343E-Mtr2 (lane 5) were analyzed by SDS-PAGE and Coomassie staining. For RNA band-shift (left panel), the indicated wild-type and mutant Mex67-Mtr2 complexes were tested in the RNA-band-shift assay using in vitro-transcribed yeast 5S rRNA. No protein (lanes 1) and increasing amounts (100 ng, 200 ng, 400 ng) of Mex67-Mtr2 (lanes 2–4), Mex67 loop KR>AA-Mtr2 (lanes 5–7), Mex67-Mtr2 RR>DD (lane 8–10), Mex67Δloop-Mtr2 (lane 11–13), and Mex67ΔloopK343E-Mtr2 (lanes 14–16). Nonshifted and band-shifted 5S rRNA are indicated. Molecular Cell 2007 26, 51-62DOI: (10.1016/j.molcel.2007.02.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Mutation of Positively Charged Amino Acids Exposed at the Loop-Confined Surface Inhibits Cell Growth (A and B) Growth dot spot analysis of the indicated mex67 (A) and mtr2 loop (B) mutants. Schematic drawing of the mutations and deletions in the loops of Mex67 and Mtr2 (upper panel). Serial dilutions of the indicated cells were spotted onto YPD plates and grown for 2 days at the indicated temperatures (lower panel). Molecular Cell 2007 26, 51-62DOI: (10.1016/j.molcel.2007.02.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Loop-Confined Surface Mutants Exhibit Specific 60S Nuclear Export Defects (A) Analysis of nuclear export of ribosomal subunits and of mRNA in the indicated mex67 and mtr2 loop mutants (the mex67-5 ts mutant was used as control to detect mRNA export defects). Subcellular location of the Rpl25-GFP (60S reporter), Rps3-GFP (40S reporter), and Nmd3-GFP (pre-60S export factor) was analyzed by fluorescence microscopy. Poly(A)+ RNA was detected by in situ hybridization with a Cy3-labeled oligo(dT) probe, and the DNA was stained with DAPI. (B) Analysis of ribosome and polysome profiles (OD254nm) of the indicated mex67 and mtr2 loop mutants by sedimentation centrifugation on sucrose density gradients. 40S, 60S, and 80S ribosomes; polysomes; and halfmer polysomes are indicated. Molecular Cell 2007 26, 51-62DOI: (10.1016/j.molcel.2007.02.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Genetic Interactions of mex67 and mtr2 Loop Mutants with the Pre-60S Export Factor Nmd3 (A) Synthetic lethality between the nmd3ΔNES1 strain combined with the indicated mutant alleles mex67Δloop, mex67 loop KR>AA, mtr2Δloop116-137, and mtr2 RR>DD. Shuffle strains nmd3Δ/mex67Δ and nmd3Δ/mtr2Δ were transformed with plasmids that carry the indicated wild-type and mutant alleles (Table S1). Transformants were spotted in 10−1 dilutions onto 5-FOA plates and incubated at 30°C for 3 days. No growth indicates synthetic lethality. (B and C) Overexpression of Mex67-Mtr2 suppresses the growth inhibition (B) and the 60S subunit export defect (C) in nmd3ΔNES mutants. (B) Shuffle strain nmd3Δ was transformed with nmd3ΔNES1 (viable) or nmd3ΔNES1+2 (not viable on 5-FOA) and indicated genes NMD3, MEX67, and MTR2 inserted into a yeast high-copy number (2 μ) plasmid (Table S2). Transformants were spotted in 10−1 dilutions onto 5-FOA plates and incubated at 30°C for 5 days. (C) Fluorescence microscopy analysis of nuclear export of 60S subunits and Nmd3 lacking its nuclear export signal (NES) under conditions of high-copy suppression by MEX67-MTR2. Subcellular location of the Rpl25-GFP and Nmd3ΔNES1-GFP in strain nmd3ΔNES1 (left panel). Subcellular location of Rpl25-GFP and Nmd3ΔNES1+2-GFP in strain nmd3ΔNES1+2 (right panel). Note that even under conditions of expression of wild-type NMD3, Nmd3ΔNES1-GFP or Nmd3ΔNES2-GFP still accumulates in the nucleus, suggesting that there is only one Nmd3 binding site per pre-60S particle. Molecular Cell 2007 26, 51-62DOI: (10.1016/j.molcel.2007.02.018) Copyright © 2007 Elsevier Inc. Terms and Conditions