Identification of SNAPIN as a New Substrate of the E3 Ubiquitin Ligase TEB4

Abstract

Nα-terminally acetylated proteins can be polyubiquitylated by a group of E3 ubiquitin ligases, termed the Ac/N-recognins. The proteolytic process is called the Ac/N-end rule pathway that represents a major biological function of Nα-terminal acetylation, one of the most common protein modifications in eukaryotes. The Ac/N-end rule pathway operates in yeast and mammals, with Doa10 and TEB4 E3 Ub ligases as the Ac/N-recognins, respectively. TEB4 is embedded in the endoplasmic reticulum (ER) membrane with 14 transmembrane domains, and its N-terminal RING domain is located in cytosolic face. Although TEB4 has recently identified as an important Ac/N-recognin in mammals, only a few binding partners for it have been discovered. In this study, I carried out a yeast two-hybrid (Y2H) screen to identify new binding partners of TEB4 using 8 cytosolic domains of this protein as baits. As a result, the SNARE-associated protein (SNAPIN) was found to interact with TEB4. SNAPIN is involved in the signal endosome transport and neurite extension. In cycloheximide (CHX)-chase assay, SNAPIN was more stabilized in small interfering RNA (siRNA)-mediated TEB4 knockdown or TEB4 (-/-) knockout cells than in wild-type HeLa cells. These results suggested that TEB4 mediates the degradation of SNAPIN, as its new substrate.