Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

Abstract

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

Figure 1.

In vitro binding of MBP-Nmd3 to 60S but not 80S ribosomes. (A) Increasing amounts of MBP-Nmd3 were incubated with Rpl25-myc–containing 60S subunits and immunoprecipitated with anti-myc antibody and protein A beads. Bound proteins were eluted in Laemmli sample buffer, separated on 12% SDS-PAGE, and stained with Coomassie brilliant blue. See Materials and methods for details. Lane 1 shows MBP-Nmd3 without 60S; lanes 2–7 show 60S-myc with increasing amounts of MBP-Nmd3 as indicated. The molar ratio of Nmd3 to 60S subunits is given for the input and bound samples. (B) Nmd3 does not bind 80S ribosomes. MBP-Nmd3 was incubated alone (lanes 1 and 2), with 60S (lanes 3 and 4), or with 80S subunits (lanes 5 and 6). Samples were layered over 60% sucrose cushions and centrifuged. Supernatants (S) and pellets (P) were separated on a 12% SDS-PAGE, and proteins were visualized by Coomassie staining. (A and B) The positions of molecular mass markers (M) are given in kilodaltons.

Figure 2.

Visualization of MBP-Nmd3 binding to the 60S subunit. (A) Intersubunit side view of the control 60S subunit. (B) Intersubunit view of the segmented 60S part of the MBP-Nmd3–60S reconstruction. Significant conformational changes are seen in the GAC, the SRL, the CP, and the region around the peptidyl-transfer center. The stalk base (sb), the L1 stalk (rpL1), and 25S rRNA helices 38, 69, and 95 (H38, H69, and H95) are also labeled. The direction of the motion of the intersubunit surface of the 60S subunit after MBP-Nmd3 binding is marked with arrows. (C) Intersubunit side view of the MBP-Nmd3–60S subunit complex. The segmented density attributed to the MBP-Nmd3 combined mass is colored red, whereas the 60S subunit is colored blue. The asterisk denotes the thread of density (see Identification of Nmd3–60S subunit interactions for details). (D) Top view of the complex showing three connections (C1, C2, and C3) of the MBP-Nmd3 mass (red) with the 60S subunit (blue).

Figure 3.

rRNA protection: MBP-Nmd3 interaction with helices H38, H65, H69, and H95 of 25S. (A) 60S subunits were incubated with no protein, MBP-Nmd3, or GST-Nmd3 and treated with RNaseV1. The rRNA was extracted, and primer extension reactions were performed to identify regions of altered sensitivity to RNaseV1. Sequencing reaction lanes are marked by the dideoxynucleotide present in the mixture. Primers used were H38, AJO1061; H65, AJO501; H69, AJO1060; and H95, AJO1135. Numbers indicate positions (E. coli numbering) of nucleotides showing major alteration in sensitivity to RNaseV1. Unp, unprotected (no Nmd3); MBP, MBP-Nmd3; GST, GST-Nmd3. (B) An interface view of the 50S subunit of the 70S E. coli crystal structure (PDB ID 2AW4) showing the position of the helices concerned. Nucleotides marked in green are protected from RNaseV1 by Nmd3; nucleotides in purple show enhancement of cleavage upon interaction with Nmd3; the nucleotide in red shows protection from RNaseV1 cleavage by GST; and the nucleotide in yellow is protected from RNaseV1 cleavage by MBP.

Figure 4.

MBP-Nmd3 interaction with the 60S subunit. (A and B) Close-up view of the quasiatomic structure of the 60S subunit (PDB ID 1S1I; Spahn et al., 2001), with the MBP-Nmd3 density showing connections with the rRNA helices (A) and nearby proteins (B). (C–E) Close-up views of the rRNA helices and the nucleotides showing altered protection patterns upon MBP-Nmd3 binding in the 50S subunit of the E. coli 70S ribosome crystal structure: H38 (C), H95 (D), and H65 and H69 (E). Nucleotides are colored as in Fig. 3 B.

Figure 5.

Schematic representation of the proposed mechanism for Nmd3 release. The Nmd3-bound 60S subunit in the cytoplasm presents a different conformation compared with the unbound 60S subunit, characterized as the result of a closing motion, as if gripping the ligand Nmd3. The usual multiple binding sites of translational GTPase factors, EF1A, eEF2, eRF3, are in the GAC region, as identified in eukaryotic and prokaryotic systems. In the present 60S complex, this region is partially blocked by the ligand. Thus, the cleft region between CP and GAC, close to the protein Rpl10 region, is a likely binding region for Lsg1. Conformational changes in Lsg1 as well as in the Rpl10-binding cleft, which accompany GTP hydrolysis, allow the 60S subunit to adopt a relaxed conformation and thus facilitate Nmd3 release. Next, the unbound 60S subunit is ready for association with the 40S initiation complex.