Volume 47, Issue 5, Pages 788-796 (September 2012) Monitoring Spatiotemporal Biogenesis of Macromolecular Assemblies by Pulse-Chase Epitope Labeling  Philipp Stelter, Ruth Kunze, Monika Radwan, Emma Thomson, Karsten Thierbach, Matthias Thoms, Ed Hurt  Molecular Cell  Volume 47, Issue 5, Pages 788-796 (September 2012) DOI: 10.1016/j.molcel.2012.06.015 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 788-796DOI: (10.1016/j.molcel.2012.06.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Design of the Translation-Controllable Pulse-Chase Technique (A–C) Construction of an mRNA encoding the ORF of interest (in this case NUP82), which can be controlled by translational induction and repression, respectively. The mRNA construct harbors a tetracycline regulatable riboswitch aptamer (tc-apta) in the 5′ UTR. After the AUG start codon comes a mini ORF encoding the HA epitope, which is terminated by an amber stop codon (UAG or stop). However, this UAG was placed in-frame to the adjacent NUP82 ORF. Finally, FPA encoding the Flag-TEV-ProtA tag was fused to the 3′ end of the NUP82 ORF to allow bait detection and affinity-purification.Translation of the mRNA in the absence of Ome-Tyr yields only HA peptides because of the presence of an amber stop codon between HA and bait ORF (A). Uptake of Ome-Tyr causes aminoacylation of the suppressor tRNAOme-Tyr, which suppresses the amber nonsense codon in the HA-UAG-NUP82-FPA mRNA, resulting in the final synthesis of the newly synthesized, full-length HA-Nup82-Flag-ProtA (B). Tetracycline binds to the tc-apta riboswitch of the engineered mRNA and, as a consequence, interferes with translation initiation (40S subunit scanning) (C). See also Figure S1A. Molecular Cell 2012 47, 788-796DOI: (10.1016/j.molcel.2012.06.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 De Novo Synthesis and Purification of the Nup82 Complex Applying the Translation-Controllable Pulse-Chase Technique (A and B) Schematic drawing (upper) of the engineered HA-UAG-NUP82-FPA mRNA without (A) and with the tetracycline aptamer (tc-apta) riboswitch (B). Western blot analysis (lower) of whole-cell lysates using anti-ProtA and anti-Arc1 antibodies to detect Ome-Tyr pulse-induced HA-Nup82-Flag-ProtA and Arc1 (loading control), respectively. The HA-UAG-NUP82-FPA construct is under the control of the GAL promoter and is present in yeast cells that also carry the tRNAOme-Tyr/tRNA-synthetase pair. Indicated from (A), lanes 2–6, are the time points of Ome-Tyr induction (pulse) in cells that were grown for 17 min in galactose. Sample before galactose induction (lane 1). Lane 7 shows the endogenous level of a Nup82-TAP strain grown for 5 hr in raffinose (steady state), but the lysate loading was 1/10 when compared to lanes 1–6. Expression of HA-Nup82-Flag-ProtA under the control of a GAL promoter and a tc-apta riboswitch is shown in (B). Lanes 2–7: Ome-Tyr induction (pulse) after 17 min GAL incubation. Lane 1: before GAL induction. Lanes 8–11 show the chase time points with tetracycline/glucose after a 3 min Ome-Tyr induction (indicated in lane 3). (C and D) Tandem affinity-purification of pulse-labeled HA-Nup82-Flag-ProtA from conditions of pulse (C) and pulse-chase (D). The final Flag peptide eluates (i.e., newly formed Nup82 complex) derived from the indicated conditions and time points were analyzed by SDS-PAGE and Coomassie-staining or western blotting using antibodies against the Flag epitope (for Nup82 detection), Dyn2 and Nup159. Loading of the chromosomal-tagged Nup82-Flag-ProtA eluates (lane 5) was ten times less when compared to lanes 1–4. The intensity of western signals was quantified using the Image QuantTL software (GE Healthcare). The indicated Nsp1, Nup159, and HA-Nup82-Flag bands were identified by mass spectrometry. See also Figure S2. Molecular Cell 2012 47, 788-796DOI: (10.1016/j.molcel.2012.06.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Analysis of Dynamic Posttranslational Protein Modification by Applying the Translational Controlled Pulse Chase Technique (A) Autoproteolytic cleavage of the nascent Nup145 precursor. Schematic drawing (upper) of the engineered HA-UAG-TAP-NUP145 mRNA with premature amber stop codon and tc-apta riboswitch. The autoproteolytic cleavage site within the Nup145 precursor yielding HA-TAP-Nup145N and Nup145C is indicated by western blot analysis (lower) of whole-cell lysates using anti-ProtA antibodies. Lanes 1+2 indicate the Ome-Tyr pulse time after 17 min galactose induction. After the 5 min Ome-Tyr pulse cells were chased with tet/glu for the indicated time points (lanes 2–7). Double input of lysate and longer exposure time of the western blot is also depicted to better reveal the nascent Nup145 chains (lanes 8–11). (B) Mannosylation of the plasma membrane protein Wsc1 during transit through the Golgi. Schematic drawing (upper) of the engineered WSC1 mRNA construct harboring the premature amber stop codon and tc-apta riboswitch for the pulse-chase assay. The UAG amber stop codon substitutes the codon corresponding proline at position 3 of the WSC1 ORF. Western blot analysis (lower) of whole-cell lysates using anti-ProtA antibodies to detect Wsc1-Flag-ProtA. Indicated are the Ome-Tyr pulse time points (lanes 1+2) after 17 min galactose induction and the chase (tet/glu) time points (lanes 3–6) after a 5 min Ome-Tyr pulse. Arc1 served as loading control. TM, transmembrane sequence; SS, signal sequence. Golgi-dependent mannosylation of Wsc1 is indicated. Molecular Cell 2012 47, 788-796DOI: (10.1016/j.molcel.2012.06.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Analysis of Preribosomal Particle Composition during 60S Ribosome Biogenesis Using the Translation-Controllable Pulse-Chase Technique (A) Scheme of 60S ribosomal subunit biogenesis depicting the dynamic association of nonribosomal factors (red and yellow) and incorporation of ribosomal (blue) proteins (Rpl) during formation and maturation of the large subunit, from the nucleolus (early), through the nucleoplasm (intermediate and late pre-60S) into the cytoplasm (mature 60S subunit). (B–D) Isolation of the evolving pre-60S ribosomal particle via tandem affinity-purification of pulse-chased HA-Rpl25-Flag-ProtA bait were analyzed by SDS-PAGE and Coomassie staining or western blotting using the antibodies against preribosomal factors and ribosomal proteins. Proteins that were identified by mass spectrometry are indicated. Cells expressing tc-apta-HA-UAG-RPL25-FPA under the GAL promoter were pulsed after 17 min galactose induction with Ome-Tyr for the indicated time points (B, lanes 1 and 2). Cells were pulsed with Ome-Tyr for 5 min (C, lane 1) or for 4 min followed by a 3 min (lane 2) and 19 min chase (lane 3) with tetracycline/glucose. Chromosomally tagged Rpl25-CBP-ProtA was also tandem affinity-purified (lane 4) and directly compared to the pulse-chase derived HA-Rpl25-Flag-ProtA eluates (lanes 1–3). Note that the Rpp0 band also contained Asc1 (a 40S subunit binding protein). Pulse-chase analysis of HA-Rpl25-Flag-ProtA isolated from cells expressing dominant-negative Rsa4 E114D-GFP. HA-Rpl25-Flag-ProtA was pulsed for 5 min with Ome-Tyr after a 30 min galactose induction (D) and was subsequently chased for 19 min with glu/tet in cells expressing GAL::RSA4-GFP (lane 1) or GAL::rsa4 E114D-GFP (lane 2). Affinity-purified HA-Rpl25-Flag-ProtA was analyzed by SDS-PAGE and Coomassie staining, and bands were identified by mass spectrometry. See also Figure S3. Molecular Cell 2012 47, 788-796DOI: (10.1016/j.molcel.2012.06.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Subcellular Location of Newly Synthesized HA-Rpl25-Flag-ProtA during Pre-60S Subunit Formation (A) Subcellular fractionation to detect newly synthesized HA-Rpl25-Flag-ProtA in cell lysates. Pulse-chase of HA-Rpl25-Flag-ProtA was performed in yeast spheroplasts. Subsequently, lysed spheroplasts (T) were separated into a crude nuclear (N) and cytosolic fraction (C). tc-apta-HA-UAG-RPL25-FPA cells were pulsed for 0 min (lanes 1–3) and 6 min (lanes 4–6) with Ome-Tyr and chased for 19 min (lanes 7–9) with tetracycline/glucose. Equivalent amounts of the fractions T, C, and N were analyzed by SDS-PAGE and western blotting using antibodies against ProtA (newly synthesized HA-Rpl25-Flag-ProtA), hexokinase (cytosolic marker), Nop1 (nucleolar marker), and Sec61 (marker for membranes, including the nuclear envelope). (B) Indirect immunofluorescence to detect newly synthesized HA-Rpl25-Flag-ProtA in fixed yeast cells. The Flag epitope on pulse-chase generated HA-Rpl25-Flag-ProtA was used to follow the subcellular location of nascent 60S subunits by indirect fluorescence microscopy, using anti-Flag primary and Alexa 488 coupled secondary antibodies. DNA was stained with DAPI. Nomarski pictures are also shown. Scale bar, 5 μm. Molecular Cell 2012 47, 788-796DOI: (10.1016/j.molcel.2012.06.015) Copyright © 2012 Elsevier Inc. Terms and Conditions