Ataxin-3 binds VCP/p97 and regulates retrotranslocation of ERAD substrates.

Expansion of a polyglutamine tract in ataxin-3 (AT3) results in spinocerebellar ataxia type 3/Machado-Joseph disease, one of the nine polyglutamine neurodegenerative diseases. Understanding the normal functions of AT3 as well as its function in the context of expansion of the polyglutamine tract is critical for understanding the disease process. AT3 is a deubiquitylating enzyme with limited information on its cellular functions. We find that transfecting cells with AT3 increases cellular levels of endoplasmic reticulum-associated degradation (ERAD) substrates, CD3delta and TCRalpha, but does not alter levels of several non-ERAD substrates. AT3 increases the level of CD3delta by decreasing its degradation; pathogenic AT3 decreases degradation to a greater extent than wild-type AT3. Knock-down of endogenous AT3 decreases levels of CD3delta, suggesting that a normal function of AT3 is to regulate levels of ERAD substrates. AT3 binds VCP/p97, a key protein responsible for extracting ERAD substrates from the ER; binding is modulated by the size of the polyglutamine tract, and mutating a sequence adjacent to the polyglutamine tract inhibits the AT3-VCP interaction and AT3-dependent accumulation of CD3delta. AT3 and Ufd1 bind VCP in a mutually exclusive manner; AT3 decreases the interaction of VCP with Ufd1 as well as with ubiquitylated proteins. Using a reconstituted system, AT3 inhibits retrotranslocation of an ERAD substrate from the ER. These data suggest that a normal function of AT3 is to regulate flow through the ERAD pathway by modulating VCP-dependent extraction of proteins from the ER.