ABSTRACT
During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3Kelch ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: A.M.H., L.C.; Methodology: A.M.H., K.M.M., J.A.G., M.C.K., L.C.; Investigation: A.M.H., K.M.M., J.A.G., M.C.K.; Resources: A.M.H., K.M.M., M.C.K.; Data curation: A.M.H.; Writing - original draft: A.M.H., K.M.M.; Writing - review & editing: A.M.H., K.M.M., L.C.; Project administration: L.C.; Funding acquisition: L.C.
Funding
This work was supported by the National Institutes of Health (R01 GM043301 and RC1 GM091791 to L.C.). Partial support for predoctoral trainees was provided by National Institutes of Health training grants (T32 GM007223 for K.M.M., T32 GM007499 for J.A.G.]. Deposited in PMC for release after 12 months.
Supplementary information
Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.169219.supplemental
- Received September 4, 2018.
- Accepted November 27, 2018.
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