1. The AAA ATPase MDN1 Acts as a SUMO-Targeted Regulator in Mammalian Pre- ribosome Remodeling 
Nithya Raman, Elisabeth Weir, Stefan Müller 
Molecular Cell 
Volume 64, Issue 3, Pages (November 2016)
DOI: /j.molcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2016 64, 607-615DOI: (10.1016/j.molcel.2016.09.039)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
MDN1 Is Physically and Functionally Associated with the Mammalian PELP1 Complex
(A) HeLa cells were transiently transfected with empty vector or FLAG-PELP1, and the FLAG-tagged proteins were immunoprecipitated using FLAG beads and analyzed for co-precipitation of endogenous MDN1 by western blotting using anti-FLAG and anti-MDN1 antibodies.
(B) HeLa cells were transiently transfected with empty vector, HA-MDN1-N (aa 1–2,379), and FLAG-PELP1 as indicated, and the FLAG-tagged proteins were immunoprecipitated using FLAG beads and analyzed for co-precipitation of HA-MDN1-N by immunoblotting with anti-FLAG and anti-HA antibodies, respectively (left panel). Scheme of MDN1 domain organization is shown. HA-MDN1-N (aa 1–2,379) covers the hexameric AAA domain and part of linker domain (right panel).
(C) HA-MDN1-N was immunoprecipitated from HeLa cells after transient transfection using HA beads, and it was analyzed for co-precipitation of endogenous PELP1 by immunoblotting using anti-HA and anti-PELP1 antibodies, respectively.
(D) HeLa cells were plated in a volume of 100 μL media in 96-well microplates (E-Plate) after transfection with siControl or siMDN1, respectively. The growth index was normalized 12 hr post-transfection, and the cell proliferation profile was monitored every hour using the RT-CES system, as detailed in the manufacturer’s protocol. The data represent the average of three experiments ± SD.
(E) HeLa cells expressing YFP-RPL27 were transfected with siControl or siMDN1, and the localization of the YFP-tagged RPL27 was monitored by immunofluorescence (left panel). The efficiency of knockdown was examined by immunoblotting using anti-MDN1 antibody, and anti-Vinculin was used as a loading control (right panel). Scale bar, 15 μm.
(F) Localization of endogenous PELP1 in HeLa cells was monitored by immunofluorescence under control conditions or upon siRNA-mediated depletion of MDN1 (left panel). Knockdown was verified by western blotting using anti-MDN1 and anti-PELP1 antibodies with anti-Tubulin antibody as a loading control (right panel). Scale bar, 10 μm.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
The Association of MDN1 with PELP1 Is Regulated by SUMO Conjugation
(A) HEK293T cell lysates were used for pull-down with GST, GST-SUMO1, or GST-SUMO2, and binding of endogenous MDN1 to the GST proteins was detected by western blotting using anti-MDN1 antibody (upper panel). The respective GST-tagged proteins were visualized by Coomassie brilliant blue (CBB) staining (lower panel).
(B) 35S-methionine-labeled HA-MDN1-N and PIAS1 were generated by in vitro transcription/translation using a rabbit reticulocyte lysate system. Proteins were used for pull-down assays using GST control or GST-SUMO2 as bait. Following separation by SDS-PAGE, proteins were detected by autoradiography (upper panel). GST-tagged bait proteins were visualized by CBB staining (lower panel).
(C) FLAG-PELP1 was immunoprecipitated from HeLa cells after transfection with siControl or siSUMO2/3, and it was probed for interaction with endogenous MDN1 by western blotting with anti-FLAG or anti-MDN1 antibodies (left panel). The efficiency of SUMO2/3 depletion was controlled by western blotting using anti-SUMO2/3 antibody with anti-Tubulin as a loading control (right panel).
(D) FLAG-tagged PELP1, PELP1-SUMO2, and PELP1K826R proteins were immunoprecipitated from HeLa cells using anti-FLAG beads, and immunoprecipitates were tested for the presence of endogenous MDN1 using anti-MDN1 antibodies.
(E) HeLa cells were transfected with empty vector, HA-MDN1-N alone, or co-transfected with FLAG-PELP1 and FLAG-PELP1-SUMO2. The FLAG-tagged proteins were captured on anti-FLAG beads, and the immunoprecipitated material was tested for association with HA-MDN1-N by anti-HA western blotting.
(F) FLAG-tagged PELP1, PELP1-SUMO2, or PELP1-SUMO2K33,35R was immunoprecipitated from HEK293T cells after transient transfection, and it was tested for the ability to bind endogenous MDN1 by immunoblotting with anti-FLAG and anti-MDN1 antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
SENP3 Regulates the Association of MDN1 and PELP1 by Controlling SUMOylation of PELP1
(A) HeLa cells were transfected with FLAG-PELP1 in control cells or cells depleted from SENP3 by siSENP3. FLAG-PELP1 was captured on anti-FLAG beads and assessed for its interaction with endogenous MDN1 by immunoblotting. Knockdown of SENP3 was validated by anti-SENP3 immunoblotting.
(B) HeLa cells were co-transfected with FLAG-PELP1 and either wild-type SENP3 or SENP3C532S as indicated. FLAG-PELP1 was immunoprecipitated and tested for interaction with endogenous MDN1 under these conditions.
(C) HeLa cells were transfected with wild-type SENP3 or SENP3C532S and their localization as well as localization of endogenous PELP1 was monitored by immunofluorescence (left panel). Equal expression of SENP3 variants and induction of PELP1 SUMOylation in SENP3C532S-expressing cells were validated by immunoblotting with anti-Vinculin as a loading control (right panel). Scale bar, 15 μm.
(D) HA-MDN1-N and FLAG-PELP1 were transfected in HeLa cells together with either SENP3 or SENP3C532S in the presence (siControl) or absence (siSUMO2/3) of SUMO2/3 paralogs. FLAG-tagged PELP1 was captured on anti-FLAG beads, and immunoprecipitates were probed for HA-MDN1-N by western blotting using anti-HA antibodies, respectively (right panel). The expression level of the proteins and the efficiency of knockdown were controlled in the input sample by immunoblotting using anti-HA, anti-FLAG, anti-SENP3, and anti-SUMO2/3 antibodies with anti-Vinculin as a loading control (left panel).
(E) HeLa cells were transfected with wild-type SENP3 or SENP3C532S under siControl or siMDN1 conditions, and the localization of PELP1 and SENP3 was monitored by immunofluorescence using anti-PELP1 and anti-SENP3 antibodies (left top and bottom panels). The efficiency of transfection and knockdown, along with PELP1 SUMOylation, was detected by immunoblotting using anti-SENP3, anti-MDN1, and anti-PELP1 antibodies with anti-Tubulin as a control (right panel). Scale bar, 15 μm.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
SENP3-Mediated DeSUMOylation of PELP1 Is Needed for Proper Pre-60S Remodeling
(A) FLAG-tagged wild-type PELP1 or PELP1K826R was transfected in HeLa cells along with wild-type SENP3 or SENP3C532S, and the FLAG-tagged proteins were analyzed for their interaction with NLE1 after IP by anti-NLE1 western blotting.
(B) The same as in (A) except FLAG-tagged proteins were examined for their interaction with endogenous MDN1.
(C) Endogenous PELP1 was immunoprecipitated from HeLa cells under control, siMDN1, or siSUMO2/3 conditions and co-expression of either wild-type SENP3 or SENP3C532S. Following IP interaction of PELP1 with RPL19, RPL27 and SENP3 were monitored using the respective antibodies.
(D) HeLa cells were transfected with wild-type SENP3 or SENP3C532S and co-stained with anti-SENP3 and anti-RPL19 antibodies to determine the localization of RPL19 under these conditions by immunofluorescence. Scale bar, 15 μm.
(E) FLAG-RPL19 was co-expressed with either wild-type SENP3 or SENP3C532S in control HeLa cells or cells depleted from MDN1 or PELP1 by siRNA. FLAG-RPL19 was captured by anti-FLAG beads, and the precipitates were analyzed for the presence of MDN1, PELP1, NLE1, and RPL27 using the respective antibodies.
(F) Model depicting the role of SUMO conjugation/deconjugation in the control of PELP1/MDN1 association and remodeling of pre-60S particles (see the Discussion for details). For simplicity the crucial contribution of MDN1 ATPase activity in the remodeling process is not shown.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions