Molting is essential for the growth and life cycle of ecdysozoa, a large group of animals that includes arthropods and nematodes. During C. elegans molting, a new cuticle is synthesized underneath the old cuticle, which is eventually perforated and discarded. Protease activity is thought to be required for the partial degradation and release of the old cuticle, as loss of specific proteases can lead to molting defects. Interestingly, peptide protease inhibitors are also necessary for nematode molting. This has led to the model that the precisely timed activation and repression of proteases is essential for normal molting. In this study we show that clathrin-mediated endocytosis (CME) promotes downregulation of the matrix metalloprotease ADM-2/ADAM9/12/19/33, a conserved member of the ADAM (a disintegrin and metalloprotease) family of transmembrane metallopeptidases. In previous work we have shown that NEKL-2/NEK8/9 and NEKL-3/NEK6/7, members of the highly conserved family of NIMA-related kinases, are required for normal molting in C. elegans. Moreover, defective molting in nekl mutants is due to defects in CME, and partial restoration of CME by mutations in the AP2 clathrin-adapter complex, also restores normal molting. Through a non-biased genetic screen, we found that loss of ADM-2 function can suppress molting defects in nekl mutants. Unlike mutations in genes associated with AP2, however, loss of ADM-2 does not correct nekl trafficking defects, indicating that suppression by
adm-2 occurs through a novel mechanism. Endogenously tagged ADM-2 is expressed in the epidermis and colocalization studies indicate that ADM-2 is trafficked via CME to the lysosome. Furthermore, loss of NEKL function leads to increased expression of ADM-2 at the epidermal plasma membrane. Importantly, CRISPR-derived mutations targeting the extracellular protease and disintegrin domains of ADM-2 strongly suppress molting defects in nekl mutants. Unlike AP2 mutations, however, loss of ADM-2 does not suppress null or strong loss-of-function nekl mutants, indicating that a failure to endocytose additional cargo, such as steroid hormone precursors required for molting, also contributes to nekl molting defects. Our findings lead to the model that excess ADM-2 activity at the plasma membrane impedes normal molting and that NEKL-mediated CME acts to restrict the activity of proteases during the molting process.