Ubiquitin-specific protease 33 (USP33) is a deubiquitinase that has been associated with a variety of physiological events. Here, we show the existence of multiple USP33 splice variants and characterize the sub-cellular localization of endogenous USP33 as well as GFP-USP33 isoforms 1-3. The localization of USP33 is broadly confined to the secretory pathway, with all variants localizing to endoplasmic reticulum-associated structures. In addition, GFP-USP33 variant 3 shows a marked accumulation at the Golgi apparatus. Several deubiquitinases have large insertions within their otherwise highly conserved catalytic domains, the function of which is poorly characterized. Analysis of USP33 reveals a role for two distinct inserts within the catalytic domain. One is required for association with the endoplasmic reticulum, whilst the second is required for membrane association, but can be alternatively spliced (variant 3) to excise eight amino acids, which otherwise suppress Golgi localization. We propose that varying the expression of differentially localized isoforms provides a means to influence the spectrum of substrates encountered by USP33.
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