The localization and putative interaction partners of UspA suggest a role in controlling

UspA-GFP is localized primarily at the nuclear periphery and subpopulations exist in the nuclei and in the cytoplasm during vegetative growth (Figure 47). BiFC and co-localization experiments with the PCI domain containing CSN subunit CsnB revealed that mostly the nuclear fraction of UspA interacts with the COP9 signalosome. The catalytic activity of UspA influences the localization slightly; the inactive mutant protein accumulates still close to nuclei and shows a population that is distributed through the cytoplasm. An A. nidulans CsnD-GFP fusion was previously observed in the cytoplasm and accumulates in nuclei (Busch et al., 2003). The catalytic active subunit Csn5/E localizes predominantly inside the nucleus when it is

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incorporated into the COP9 signalosome (Tomoda et al., 2002). Single Csn5/E or smaller subcomplexes were also detected in the cytoplasm. Csn5 carries an NES, which serves as recognition factor for the export factor CRM1 (Tomoda et al., 2002).

The human Usp15 and its paralogous proteins Usp11 and Usp4 are all at least partially localized inside nuclei (Sowa et al., 2009; Vlasschaert et al., 2015). Usp4 is mainly localized in the nucleus, but excluded from the nucleolus, whereas its paralog Usp15 shows preferably cytoplasmic or nucleolus localization in NIH3T3 or HeLa cells (Soboleva et al., 2005).

Consistently, the S. pombe ortholog Ubp12 has a cytoplasmic and nuclear subpopulation (Kouranti et al., 2010). The ubiquitin-specific protease UBP109 is the rat ortholog of human Usp15 (Park et al., 2000). Three nuclear localization sequences were recognized in its sequence, whereas only one influences subcellular localization (Park et al., 2000). This NLS motif with the sequence – LKKR – is also present in human Usp15. In silico analysis of the amino acid sequence of UspA with the NucPred Software was performed, which calculates the likelihood for every single amino acid to be involved in nuclear localization (Brameier et al., 2007). It revealed a NLS with very high scores for the amino acid sequence “RRKKK”, a region close to its C-terminus spanning residues 1034-1038. This region is identified as potential NLS also with the software cNLS Mapper with a score of 3.5 (Kosugi et al., 2009). This score is rather low and means that the protein is localized in both, cytoplasm and nucleus. This result fits well to the observed localization pattern of UspA in A. nidulans. UspA-GFP as well as UspA^AA-GFP pull numerous proteins related to nuclear transport, like the kayopherins KapB and KapF (Figure 47).

Whereas KapF is localized in nuclei, KapB was observed at the nuclear periphery and both proteins are essential for fungal life (Markina-Inarrairaegui et al., 2011). Nucleocytoplasmic shuttling activity was attributed to the Usp4 ortholog in mice (Soboleva et al., 2005). Mice Usp4 contains NLS as well as NES sequences and migrates into the nucleus with the importin α/β. In general, DUBs can be localized in the nucleus or in the cytoplasm and can regulate nuclear transport of proteins. This study provides evidence that in the filamentous fungus A. nidulans, where several nuclei can be part of one cellular compartement, the deubiquitinase UspA is pimarily localized in close proximity to nuclei. Due to its putative NLS and putative interaction partners that are involved in nuclear transport, it is reasonable to suggest a function of the deubiquitinase in nuclear transport.

Besides the localization of DUBs itself, they can be involved in the regulation of subcellular localization of other proteins by removing ubiquitination patterns of certain lysine residues in the target protein. The subcellular localization of the phosphatase and tensin homolog (PTEN), which functions as tumor suppressor, is influenced by monoubiquitination events in cancer cell

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lines (Trotman et al., 2007). Furthermore, deubiquitination of PTEN ensures the destination inside the nucleus. Deubiquitinated PTEN is supposed to perform its function in the nucleus, whereas ubiquitinated forms will be shuttled to the cytoplasm. In this example, ubiquitination of certain lysine residues directly affects cellular localization (Trotman et al., 2007). It is possible that UspA is involved in such a regulatory mechanism where the substrates of deubiquitination are still unknown.

Figure 47: UspA accumulates primarily close to nuclei and appears in smaller subpopulations inside the nucleus as well as in the cytoplasm.

UspA removes ubiquitin (Ub) molecules from substrates (S) and accumulates close to nuclei. It mainly interacts with subunits of the COP9 signalosome inside the nucleus, but a subpopulation is also located at the nuclear periphery. UspA interacts with nuclear import factors, which might recognize the putative NLS located in the C-terminal part of UspA and might be involved in translocation of the deubiquitinase into the nucleus.

Furthermore, the active and the inactive UspA fusion protein versions pulled eleven proteins related to transcriptional processing altogether. Pull downs of the UspA protein suggest a function of the deubiquitinase in transcriptional elongation as the active UspA-GFP pulled two Polymerase II transcription elongation factors and two proteins, which regulate the transcription.

The inactive UspA^AA-GFP pulled a putative subunit of the Ccr4-Not complex. BLAST analyses revealed that the pulled protein AN4965 has highest similarities with the subunit Not1 of this protein complex, the scaffolding protein. This Ccr4-Not1 complex has several functions in gene transcription and translation processes (Collart, 2016). Usp4 and Usp15 were suggested to have

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a role in RNA processing in humans as they associate to members of the splicing machinery (Sowa et al., 2009). Usp11 rather associates to different transcription elongation factors (Sowa et al., 2009).

Taken together, the function of fungal and mammalian USP orthologs is quite conserved. Usp15, Usp4 and Usp11 interact with proteins related to transcription and translation processes or with proteins that are involved in RNA processing steps (Song et al., 2010; Sowa et al., 2009). The A. nidulans ortholog UspA pulls several proteins related to this processes in GFP pull down experiments as well.

4.6 UspA regulates the protein abundance of the major developmental regulator VeA and