Abstract
Endoplasmic reticulum (ER) tubules connect each other by three-way junctions, resulting in a tubular ER network. Oligomerization of three-way junction protein lunapark (Lnp) is important for its localization and the three-way junction stability. On the other hand, Lnp has an N-terminal ubiquitin ligase activity domain, which is also important for the three-way junction localization. To understand the mode of action of Lnp, we isolated Cullin-associated and neddylation-dissociated 1 (CAND1), a regulator of Skp1-Cul1-F-box (SCF) ubiquitin ligase, as a Lnp-binding protein by affinity chromatography. CAND1 and Lnp form a higher-molecular-weight complex in vitro, while they do not co-localize at the three-way junctions. CAND1 reduces the auto-ubiquitination activity of Lnp. CAND1 knockdown enhances proteasomal degradation of Lnp and reduces the tubular ER network in mammalian cells. These results suggest that CAND1 has the potency to promote the formation of the higher-molecular-weight complex with Lnp and reduce the auto-ubiquitination activity of Lnp, thereby regulating the three-way junction stability of the tubular ER network.