The small protein ubiquitin is an essential post-translational modification that regulates a vast number of cellular processes in eukaryotes. Through a cascade of enzymatic steps, ubiquitin is transferred from a ubiquitin activating enzyme (E1) to a ubiquitin conjugating enzyme (E2), which interacts with a ubiquitin ligase (E3) that stimulates transfer of ubiquitin to a substrate. Conjugation of ubiquitin to a substrate can result in a variety of outcomes, including changes in protein stability, localization, or binding partners. This modification is utilized to control a diversity of processes, including protein trafficking, cell division, DNA damage response, immune signaling, transcriptional regulation, and many others.

Cullin-RING-ligases (CRLs) are the largest class of ubiquitin E3 ligases. CRLs are comprised of one of seven Cullin scaffold proteins (CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and CUL7). The functions of these ligases are nearly as diverse as the function of ubiquitin itself. In particular, CUL3 has functions in developmental processes, mitosis, autophagy, organelle formation, endocytosis, and more. CUL3 exerts its many roles by pairing with at least 90 substrate receptors that recruit proteins to be ubiquitylated. When in complex with the substrate receptor KLHL12, CUL3 transfers a single ubiquitin to its substrate, SEC31, a structural component of the COPII vesicle coat that helps transport proteins from the endoplasmic reticulum to the Golgi apparatus. SEC31 ubiquitylation triggers an increase in COPII vesicle size, allowing for the packaging and transport of large proteins such as collagen.

In this work, I examine the mechanisms by which SEC31 ubiquitylation by CUL3KLHL12 is regulated. CUL3KLHL12 activity requires the calcium-binding proteins PEF1 and ALG2. PEF1 and ALG2 form a target-specific co-adaptor that acts as a calcium sensor. Together PEF1 and ALG2 are an integral part of the CUL3KLHL12 ubiquitin ligase and allow for SEC31 targeting only in the presence of elevated cellular calcium concentrations. This translates a short-lived rise in cytosolic calcium levels into sustained ubiquitylation of SEC31, which in turn triggers formation of large COPII coats and collagen secretion. As calcium signaling is also known to control chondrocyte differentiation and collagen synthesis, the regulated activation of CUL3KLHL12 could allow organisms to integrate collagen secretion into programs of craniofacial bone formation. This work shows that target-specific co-adaptors provide a mechanism for rapid and signal-dependent activation of CUL3 complexes important for human development and disease.