Posttranslational modifications govern protein function by modulating their structure, localization, dynamics, and/or stability. Ubiquitination of substrate proteins induces an array of specific responses depending on the extent and architecture of the modification. Proteins can be modified by addition of a single ubiquitin on a single site (monoubiquitination) or multiple sites (multiple monoubiquitination) or by polymerization of ubiquitin monomers into chains of specific linkages (polyubiquitination) 
. Specific ubiquitin configurations elicit unique cellular responses and affect essential processes including protein degradation, DNA repair, chromatin remodeling, endocytosis, and cell cycle regulation 
. Due to the vital roles of ubiquitination, this process is highly regulated and requires a cascade of three enzymes, culminating in a substrate- and site-specific modification 
. Likewise, cleavage of ubiquitin moieties or chains by deubiquitinating enzymes (DUBs) must be tightly regulated in space and time 
DUBs are highly conserved cysteine proteases or metalloproteases that can be classified based on their catalytic domain structure: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), Machado-Joseph disease proteases, and JAB1/MPN/Mov34 metalloenzymes (JAMMs) 
. The diversity of DUB catalytic core and domain structures, as well as their number (approximately 95 DUBs encoded by the human genome), reflects their involvement in multiple essential roles including (1) processing of ubiquitin precursor proteins, (2) recycling of ubiquitin trapped in modified, inactivatable forms, (3) cleavage of ubiquitin from target proteins, and (4) regeneration of monoubiquitin from free polyubiquitin chains 
Specific functions of several DUBs have been elucidated. A trio of DUBs (Rpn11/PSMD14, Uch2/UCHL5, and Ubp6/USP14) act at the proteasome to remodel or remove ubiquitin chains prior to substrate degradation 
. Other DUBs play roles in transcriptional regulation (Ubp8p/USP22), downregulation of the NFκB pathway (CYLD), DNA repair (USP1), or membrane trafficking between the endoplasmic reticulum (ER) and the Golgi complex (Ubp3p) 
. Although a role for DUBs in several pathways has been defined, their enzymatic targets and modes of regulation remain largely unknown 
. A recent proteomic study of human DUBs assigned potential roles to previously uncharacterized DUBs by placing them in putative cellular contexts defined mainly by the nature of their interactors 
. However, despite such efforts to link various DUBs to different cellular functions in several organisms, there are still significant gaps in our understanding of the action and regulation of these enzymes.
In this study, we characterize the entire family of DUBs in the fission yeast Schizosaccharomyces pombe.
In contrast to mammalian cells, the S. pombe
genome encodes only 20 putative DUBs belonging to four of the five DUB subfamilies (UCH, USP, OTU, and JAMM; ). A handful of other proteins in the S. pombe
genome encode DUB domains (Ubp10, Ubp13, Rpn8, Csn5, Cwf6, eIF3f, and eIF3h), but they are either lacking the full complement of catalytic residues necessary for protease function (Figure S1
) or, in the case of the signalosome component Csn5, have activity towards other ubiquitin-like proteins and have been excluded from our consideration 
. All S. pombe
DUBs are nonessential for viability, except for one of the proteasomal DUBs, Rpn11 
. We chose to study the S. pombe
DUB family because of the limited number of DUBs encoded by this genome, the conservation of catalytic core structures and some non-catalytic domain modules () 
, and the genetic tractability of S. pombe,
which allows endogenous gene tagging and simple genetic manipulation. These attributes confer a significant advantage for a genome-wide study and the potential to comprehensively assign DUB activities to functional networks.
Inventory and domain architecture of S. pombe DUBs.
We took a multifaceted approach to investigate S. pombe
DUBs, combining the determination of endogenous localizations, evaluation of their in vitro activity, and proteomic analysis of protein interactions. To our knowledge, this work provides the first systematic localization study of a complete DUB family and reveals that S. pombe
DUBs are present in nearly every cellular compartment. Moreover, our proteomic approach identified stable protein–protein interactions for over 55% of the S. pombe
DUBs. By means of subcellular localization studies and activity assays we show how three uncharacterized DUBs are regulated by non-catalytic partners, including a potential interactor for human USP7/HAUSP, which controls the tumor suppressor p53 
. We also found that a conserved DUB complex participates in endocytosis, actin organization, and cell polarity and that these cellular functions are shared by at least five different DUBs. The powerful combination of experimental approaches utilized in this study reveals new examples of regulation for this important protein family.