UBPY Controls the Stability of ESCRT-0 Complex in Development. Junzheng Zhang, Ying Su, Min Liu, Alan Jian Zhu. Dept. of Cellular & Molecular Medicine, Lerner Res Inst, Cleveland Clinic, Cleveland, OH.

   Ubiquitinated developmental signaling proteins are often internalized and then sorted into the endocytic trafficking pathway for degradation. The deubiquiting enzyme UBPY is believed to prevent protein degradation by removing ubiquitin modifier from internalized signaling protein cargos. This protective role of UBPY is consistent with recent Drosophila studies of endocytosed Frizzled2 and Smoothened. However, this canonical model is challenged by several studies on USP8, the vertebrate homolog of UBPY, in which USP8 promotes endocytosed cargo degradation. Here, we utilize both RNAi and loss of function allele of Ubpy to demonstrate that UBPY is not required for protecting cargo from degradation in vivo. Instead, in the absence of Ubpy, a panel of signaling proteins important for Drosophila development accumulates in enlarged Rab5-positive endosomes, a phenotype that is often associated with defects in the ESCRT (endosomal sorting complex required for transport) complexes. Indeed, we find that UBPY directly interacts with and deubiquitinates Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate), an essential component of the ESCRT-0 complex. Furthermore, the activity of the ESCRT-0 complex is compromised in Ubpy-null cells where ubiquitinated Hrs undergoes lysosome-mediated degradation. Consistently, we find that developmental signaling proteins are enriched in early endosomes when hrs activity is abolished in Drosophila. Finally, we provide evidence that USP8 stabilizes Hrs in vertebrate cells. Thus, our study uncovers a critical role of UBPY in protecting the ESCRT-0 complex from degradation, a control mechanism in degradative protein sorting that is also conserved in higher organisms.