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The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

Gerhardt C, Lier JM, Burmühl S, Struchtrup A, Deutschmann K, Vetter M, Leu T, Reeg S, Grune T, Rüther U - J. Cell Biol. (2015)

Bottom Line: Mutations in RPGRIP1L result in severe human diseases called ciliopathies.Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l.We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany Christoph.Gerhardt@hhu.de.

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Rpgrip1l interacts with the 19S proteasomal subunit component Psmd2. (A) Coimmunoprecipitation experiments in NIH3T3 cells. Myc-tagged Psmd2 full-length protein and FLAG-tagged RID were transiently overexpressed and tested for interaction by coimmunoprecipitation from total cell lysates. Immunoprecipitation assays were performed by using an anti-FLAG antibody. Myc-Psmd2 coimmunoprecipitated with FLAG-RID (lane 3). Ctrl., control; IP, immunoprecipitation; WB, Western blot. (B) Myc-tagged Psmd2 full-length protein and FLAG/Strep/HA-tagged RID domain of Rpgrip1l were transiently overexpressed in HEK293T cells and tested for interaction by tandem affinity purification (TAP) tag experiments from total cell lysates. FLAG/Strep/HA-tagged RID was immunopurified using anti-FLAG beads, eluted with FLAG-protein, and recaptured on anti-Strep beads. The final eluation was performed by using biotin. After this sequential purification, we identified Myc-Psmd2 (lane 2) but not the unrelated protein Gatad1 (lane 6) in addition to purified FLAG/Strep/HA-RID. (C–E) Immunofluorescence on MEFs isolated from WT or Rpgrip1l−/− E12.5 embryos (n = 3 embryos, respectively). (C) Centrosomes are marked by γ-tubulin as well as acetylated α-tubulin (α-Tub), and cell nuclei were marked by DAPI. Insets illustrate higher magnifications. (D) The ciliary axoneme and the BB are marked by acetylated α-tubulin and by γ-tubulin, respectively. The plot shows fluorescence intensities of the depicted representative image. (E) In situ proximity ligation assay (in situ PLA) on MEFs. Cell nuclei are marked by DAPI, and the ciliary axoneme are marked by transiently transfected Ift88-EYFP. Additional accumulation of Ift88-EYFP at the ciliary base is highlighted by yellow brackets. White arrowheads point to cilia. Bars: (C, all images; and D, overview) 10 µm; (D and E, magnifications) 1 µm.
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fig7: Rpgrip1l interacts with the 19S proteasomal subunit component Psmd2. (A) Coimmunoprecipitation experiments in NIH3T3 cells. Myc-tagged Psmd2 full-length protein and FLAG-tagged RID were transiently overexpressed and tested for interaction by coimmunoprecipitation from total cell lysates. Immunoprecipitation assays were performed by using an anti-FLAG antibody. Myc-Psmd2 coimmunoprecipitated with FLAG-RID (lane 3). Ctrl., control; IP, immunoprecipitation; WB, Western blot. (B) Myc-tagged Psmd2 full-length protein and FLAG/Strep/HA-tagged RID domain of Rpgrip1l were transiently overexpressed in HEK293T cells and tested for interaction by tandem affinity purification (TAP) tag experiments from total cell lysates. FLAG/Strep/HA-tagged RID was immunopurified using anti-FLAG beads, eluted with FLAG-protein, and recaptured on anti-Strep beads. The final eluation was performed by using biotin. After this sequential purification, we identified Myc-Psmd2 (lane 2) but not the unrelated protein Gatad1 (lane 6) in addition to purified FLAG/Strep/HA-RID. (C–E) Immunofluorescence on MEFs isolated from WT or Rpgrip1l−/− E12.5 embryos (n = 3 embryos, respectively). (C) Centrosomes are marked by γ-tubulin as well as acetylated α-tubulin (α-Tub), and cell nuclei were marked by DAPI. Insets illustrate higher magnifications. (D) The ciliary axoneme and the BB are marked by acetylated α-tubulin and by γ-tubulin, respectively. The plot shows fluorescence intensities of the depicted representative image. (E) In situ proximity ligation assay (in situ PLA) on MEFs. Cell nuclei are marked by DAPI, and the ciliary axoneme are marked by transiently transfected Ift88-EYFP. Additional accumulation of Ift88-EYFP at the ciliary base is highlighted by yellow brackets. White arrowheads point to cilia. Bars: (C, all images; and D, overview) 10 µm; (D and E, magnifications) 1 µm.

Mentions: To unravel how Rpgrip1l regulates the activity of the proteasome, we searched for novel interaction partners of Rpgrip1l by performing a yeast two-hybrid screen. Among a total of six proteins, we found Psmd2 as a potential interaction partner (Fig. S1 C). We used the tagged RPGR-interacting domain (RID) of Rpgrip1l (the full-length Rpgrip1l could not be stably expressed) and a tagged full-length Psmd2 for transient overexpression in NIH3T3 and HEK293T cells. Using coimmunoprecipitation and tandem affinity purification assays, we confirmed the interaction of Rpgrip1l with Psmd2 (Fig. 7, A and B).


The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

Gerhardt C, Lier JM, Burmühl S, Struchtrup A, Deutschmann K, Vetter M, Leu T, Reeg S, Grune T, Rüther U - J. Cell Biol. (2015)

Rpgrip1l interacts with the 19S proteasomal subunit component Psmd2. (A) Coimmunoprecipitation experiments in NIH3T3 cells. Myc-tagged Psmd2 full-length protein and FLAG-tagged RID were transiently overexpressed and tested for interaction by coimmunoprecipitation from total cell lysates. Immunoprecipitation assays were performed by using an anti-FLAG antibody. Myc-Psmd2 coimmunoprecipitated with FLAG-RID (lane 3). Ctrl., control; IP, immunoprecipitation; WB, Western blot. (B) Myc-tagged Psmd2 full-length protein and FLAG/Strep/HA-tagged RID domain of Rpgrip1l were transiently overexpressed in HEK293T cells and tested for interaction by tandem affinity purification (TAP) tag experiments from total cell lysates. FLAG/Strep/HA-tagged RID was immunopurified using anti-FLAG beads, eluted with FLAG-protein, and recaptured on anti-Strep beads. The final eluation was performed by using biotin. After this sequential purification, we identified Myc-Psmd2 (lane 2) but not the unrelated protein Gatad1 (lane 6) in addition to purified FLAG/Strep/HA-RID. (C–E) Immunofluorescence on MEFs isolated from WT or Rpgrip1l−/− E12.5 embryos (n = 3 embryos, respectively). (C) Centrosomes are marked by γ-tubulin as well as acetylated α-tubulin (α-Tub), and cell nuclei were marked by DAPI. Insets illustrate higher magnifications. (D) The ciliary axoneme and the BB are marked by acetylated α-tubulin and by γ-tubulin, respectively. The plot shows fluorescence intensities of the depicted representative image. (E) In situ proximity ligation assay (in situ PLA) on MEFs. Cell nuclei are marked by DAPI, and the ciliary axoneme are marked by transiently transfected Ift88-EYFP. Additional accumulation of Ift88-EYFP at the ciliary base is highlighted by yellow brackets. White arrowheads point to cilia. Bars: (C, all images; and D, overview) 10 µm; (D and E, magnifications) 1 µm.
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fig7: Rpgrip1l interacts with the 19S proteasomal subunit component Psmd2. (A) Coimmunoprecipitation experiments in NIH3T3 cells. Myc-tagged Psmd2 full-length protein and FLAG-tagged RID were transiently overexpressed and tested for interaction by coimmunoprecipitation from total cell lysates. Immunoprecipitation assays were performed by using an anti-FLAG antibody. Myc-Psmd2 coimmunoprecipitated with FLAG-RID (lane 3). Ctrl., control; IP, immunoprecipitation; WB, Western blot. (B) Myc-tagged Psmd2 full-length protein and FLAG/Strep/HA-tagged RID domain of Rpgrip1l were transiently overexpressed in HEK293T cells and tested for interaction by tandem affinity purification (TAP) tag experiments from total cell lysates. FLAG/Strep/HA-tagged RID was immunopurified using anti-FLAG beads, eluted with FLAG-protein, and recaptured on anti-Strep beads. The final eluation was performed by using biotin. After this sequential purification, we identified Myc-Psmd2 (lane 2) but not the unrelated protein Gatad1 (lane 6) in addition to purified FLAG/Strep/HA-RID. (C–E) Immunofluorescence on MEFs isolated from WT or Rpgrip1l−/− E12.5 embryos (n = 3 embryos, respectively). (C) Centrosomes are marked by γ-tubulin as well as acetylated α-tubulin (α-Tub), and cell nuclei were marked by DAPI. Insets illustrate higher magnifications. (D) The ciliary axoneme and the BB are marked by acetylated α-tubulin and by γ-tubulin, respectively. The plot shows fluorescence intensities of the depicted representative image. (E) In situ proximity ligation assay (in situ PLA) on MEFs. Cell nuclei are marked by DAPI, and the ciliary axoneme are marked by transiently transfected Ift88-EYFP. Additional accumulation of Ift88-EYFP at the ciliary base is highlighted by yellow brackets. White arrowheads point to cilia. Bars: (C, all images; and D, overview) 10 µm; (D and E, magnifications) 1 µm.
Mentions: To unravel how Rpgrip1l regulates the activity of the proteasome, we searched for novel interaction partners of Rpgrip1l by performing a yeast two-hybrid screen. Among a total of six proteins, we found Psmd2 as a potential interaction partner (Fig. S1 C). We used the tagged RPGR-interacting domain (RID) of Rpgrip1l (the full-length Rpgrip1l could not be stably expressed) and a tagged full-length Psmd2 for transient overexpression in NIH3T3 and HEK293T cells. Using coimmunoprecipitation and tandem affinity purification assays, we confirmed the interaction of Rpgrip1l with Psmd2 (Fig. 7, A and B).

Bottom Line: Mutations in RPGRIP1L result in severe human diseases called ciliopathies.Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l.We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany Christoph.Gerhardt@hhu.de.

Show MeSH
Related in: MedlinePlus