The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.
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.
Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany Christoph.Gerhardt@hhu.de.Show MeSH
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Mentions: Because in MEFs, the interaction of Rpgrip1l and Psmd2 seems to take place only at the ciliary TZ, but Rpgrip1l is also located at centrosomes during mitosis, the question arises whether Rpgrip1l also regulates the activity of the centrosomal proteasome. Proteasomal degradation at the centrosome has previously been reported for ubiquitinated proteins (Fabunmi et al., 2000; Puram et al., 2013). Using MG132, an inhibitor of proteasomal activity, we were able to show that the centrosomal (pericentriolar) proteasome is also responsible for the degradation of phospho-(S33/37/T41)-β-Catenin (Fig. 8 A). In contrast to the situation at the ciliary base, the amount of phospho-(S33/37/T41)-β-Catenin and ubiquitinated proteins is unaltered at the centrosomes of Rpgrip1l−/− MEFs (Fig. 8, B and C). These results correspond to the finding that the green fluorescence signal of the Proteasome Sensor Vector exclusively accumulates at the ciliary base in the absence of Rpgrip1l (Fig. 4 L), indicating that Rpgrip1l only affects the activity of the ciliary proteasome in MEFs. Consequently, our data reveal a regulation of proteasomal activity by Rpgrip1l exclusively at primary cilia and most likely via the interaction with Psmd2. To evaluate whether Rpgrip1l deficiency affects the activity of the 26S proteasome by affecting the function of the 19S proteasomal subunit, the amount of ubiquitinated proteins was determined at the ciliary base of MEFs in which several components of the 19S proteasomal subunit (Psmd2, Psmd3, or Psmd4) were knocked down. In MEFs, Psmd2, Psmd3, and Psmd4 knockdowns result in a significantly higher amount of ubiquitinated proteins at the ciliary base (Fig. 9, A, B, D, and F; and Fig. S2, A and B), mimicking the Rpgrip1l−/− status. This result suggests that Rpgrip1l regulates the activity of the ciliary proteasome by affecting the 19S proteasomal subunit. Remarkably, knockdowns of all these 19S proteasomal subunit components lead to significant ciliary elongation in MEFs (Fig. 9, C, E, and G). Considering that Rpgrip1l interacts with Psmd2 by means of its RID domain, we tested whether Rpgrip1l overexpression increases the activity of the ciliary proteasome by transfecting the RID domain of Rpgrip1l into NIH3T3 cells as well as into WT MEFs. We quantified the amount of Ubiquitin at the ciliary base as well as ciliary length. The amount of Ubiquitin was significantly decreased at the ciliary base of RID-transfected NIH3T3 cells and WT MEFs (Fig. 10, A and C), indicating that the ciliary proteasome is regulated by Rpgrip1l via its interaction with Psmd2 at the ciliary TZ. Interestingly, cilia were significantly longer after RID transfection (Fig. 10, B and D). To exclude that these effects are caused by an overexpression of any type of protein, we also transfected a control protein (Wnk2) into NIH3T3 cells and detected no alteration in Ubiquitin amount or cilia length (Fig. S2, C and D). Because the transfection of the RID domain leads to an increased activity of the ciliary proteasome, we also transfected the RID domain into Rpgrip1l−/− MEFs to elevate the activity of the ciliary proteasome. Indeed, the ciliary amount of Ubiquitin was restored in these MEFs (Fig. 10 C). However, ciliary length of Rpgrip1l−/− MEFs was unaltered after transfection (Fig. 10 D), implying that the RID domain of Rpgrip1l is essential for the regulation of the ciliary proteasome but is dispensable for the regulation of cilia length. Consequently, another part of the Rpgrip1l protein is needed to control the length of cilia.
Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany Christoph.Gerhardt@hhu.de.