3. Results
3.5 UspA interacts with proteins involved in nuclear transport, RNA processing and the
GFP pull down experiments were performed to identify interaction partners of UspA including putative substrates for deubiquitination. Due to the velocity of catalytic reactions, like deubiquitination processes, GFP pull downs were additionally performed with the UspAAA-GFP mutant protein. The mutated protein might have an increased binding affinity to its substrates than the functional UspA-GFP fusion protein as it cannot catalyze the enzymatic reaction. A strain overexpressing free GFP served as negative control. GFP pull down experiments were performed with crude cell extracts of cultures grown for 20 h under submerged conditions. The functional as well as the mutant fusion protein could be identified in the elution fractions of the GFP pull downs using western hybridization experiments with a αGFP antibody (Figure 29).
Figure 29: UspA-GFP and UspAAA-GFP were enriched in GFP pull down experiments.
Western hybridization experiments were performed with aliquots of different GFP pull down elution fractions (E1, E2). Signals for UspA-GFP as well as for UspAAA-GFP fusion protein were observed. Protein concentrations were higher in the first elution (E1) than in the second (E2).
The red arrow marks the respective UspA-GFP fusion protein signal, degradation products and unspecific signals are visible below.
Protein compositions of the elution fractions were analyzed with LC/MS-MS. Proteins that were identified in at least two out of three biological replicates with a log2 LFQ intensity equal to or greater than 21 and that were not identified in the GFP control were further considered for data analysis. In total, 59 proteins were used for further data analysis (Figure 30, Tables 14-16).
Among them, 38 proteins were pulled by both fusion proteins: UspA-GFP and UspAAA-GFP (Table 14, Figure 30). Furthermore, 14 proteins were only pulled down by UspAAA-GFP (Table 15, Figure 30), whereas seven proteins were only identified in the pull downs of the functional UspA-GFP (Table 16, Figure 30).
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Figure 30: Identification of putative interaction partners of UspA-GFP and UspAAA-GFP with LC/MS-MS.
The heat map displays the log2 label free quantification (LFQ) intensities of proteins identified in GFP pull down experiments. The results of three independent experiments of GFP control, UspA-GFP and UspAAA-GFP are shown. The identified proteins were assigned to different categories that are illustrated as pie charts. The numbers represent the amount of proteins assigned to the different classes. Data analysis was performed with the MaxQuant and Perseus software.
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The bait protein UspA was identified with highest intensity values and highest amount of unique peptides in pull downs with both fusion proteins. Among the 38 proteins, which were identified in the pull downs of both protein variants, one third was assigned to the group of primary metabolism (Table 14). Two members of the Zomes were identified. The Zomes comprise the three structural very similar protein complexes COP9 signalosome, the proteasomal LID and the eukaryotic initiation factor 3 (eIF3) (Pick and Pintard, 2009). Both UspA versions pulled one subunit of the proteasomal LID, RpnF, and one subunit of eIF3, eIF3D. Furthermore, five proteins related to nuclear transport, including the karyopherins KapB (AN0906) and KapF (AN6734) were pulled. Proteins related to transcriptional processing and fungal development were also detected.
Table 14: Functional groups of proteins identified with LC/MS-MS in pull downs of UspA-GFP and UspAAA-GFP.
Description of identified proteins derive from information on AspGD or FungiDB (Cerqueira et al., 2014; Stajich et al., 2012). Domain predictions for uncharacterized proteins were performed with NCBI CD domain prediction tool (Marchler-Bauer et al., 2015).
Systematic
Name Description
Primary metabolism AN3524 NAD binding Rossmann fold oxidoreductase AN7199 Uncharacterized protein, galactonate metabolism
AN0723 Uncharacterized protein, domains of sulfotransfer superfamily AN7590 Uncharacterized protein, mannitol dehydrogenase AN7895 Zinc-binding alcohol dehydrogenase domain-containing protein, CipB AN1023 Actin cytoskeleton-regulatory complex protein, End3
AN3877 Uncharacterized, domains of NTF2 like superfamily AN6734 KapF
AN5376 Uncharacterized, domains of NTF2 like superfamily
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Systematic
Name Description
Nuclear transport
AN6978 RCC1, chromatin associated guanine nucleotide exchange factor for Ran Development
AN10311 Cell wall mannoprotein, MnpA AN5635 Neutral trehalase
AN2523 Chitin synthase B
AN6709 Guanyl-nucleotide exchange factor (Sec7), hyphal morphogenesis Transcriptional processing
AN5452 Pre-mRNA-splicing factor, Rse1
AN1205 Prefoldin subunit 5, regulation of transcriptional elongation AN5894 Pol II transcription elongation factor subunit Cdc73
Ubiquitin-proteasome system AN6354 Ubiquitin carboxyl-terminal hydrolase, UspA AN7422 Ubiquitin carboxyl-terminal hydrolase
Zomes
AN7540 Eukaryotic translation initiation factor 3 subunit D, eIF3D AN10519 Proteasome regulatory particle subunit, RpnF
Signaling
AN1545 Protein phosphatase PP2A regulatory subunit B AN12477 Uncharacterized protein, GTPase activity
AN10691 Dynamin GTPase
Uncharacterized proteins AN0860 Uncharacterized protein
AN3121 Uncharacterized, conserved glutamic acid-rich protein AN10518 Uncharacterized protein
AN2647 Uncharacterized protein
AN3709 Uncharacterized protein, CRAL/TRIO domain protein
The putative USP AN7422 was coenriched with UspA. This protein was found in BLAST analyses of human Usp15 against the A. nidulans database (Table 13). qRT-PCRs were performed to investigate if the transcript levels are similarly upregulated in ΔcsnE as for uspA and the other putative USPs (Figure 21, Figure 31). The transcript levels of AN7422 were upregulated during multicellular fungal development, but not during vegetative growth, in the
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absence of CsnE. This is similar to the gene expression profiles of the other ubiquitin-specific protease encoding genes in a ΔcsnE strain (Figure 21).
Figure 31: AN7422 transcript levels are upregulated in the absence of functional COP9 signalosome similar to uspA.
Expression levels of AN6354 (uspA) and AN7422 were analyzed using qRT-PCR. RNA derived from mycelia grown for 20 h in submerged culture (veg), from asexually (asex) or sexually (sex) grown mycelia were used. As reference genes served h2A and 15S rRNA. Wild type expression was set to 1. Error bars represent standard error of the mean (SEM) of at least two biological replicates.
UspAAA-GFP pull downs were performed to identify substrates of the deubiquitinase. 14 proteins were identified in at least two out of three biological replicates only in the strain expressing the UspAAA-GFP mutant (Table 15). UspAAA-GFP might bind longer to its putative substrates as it cannot catalyze the deubiquitination reaction. Identification of polyubiquitin in the pull downs with the mutated UspA-GFP version suggests that UspA does not bind to its substrates directly, but associates to their polyubiquitin chains.
Most proteins specifically pulled down by the inactive UspA-GFP mutant were assigned to primary metabolism, but also four proteins related to transcriptional processing were identified (Table 15). PhiA, a protein involved in fungal asexual development and a serine/threonine kinase were pulled down as well. UspA-GFP, but not UspAAA-GFP, pulled four more proteins categorized to transcriptional processing and three classified to primary metabolism (Figure 30, Table 16).
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Table 15: Proteins that were identified only in the pull down of UspAAA-GFP, but not in the pull down with functional UspA-GFP fusion protein.
Description of identified proteins derive from information on AspGD or FungiDB (Cerqueira et al., 2014; Stajich et al., 2012). Domain predictions for uncharacterized proteins were performed with NCBI CD domain prediction tool (Marchler-Bauer et al., 2015).
Systematic
AN7680 Uncharacterized protein, SMC superfamily, structural maintenance of chromosomes
AN0646 DNA/RNA helicase activity
AN2007 Small nuclear ribonuceloprotein SmD3, mRNA binding AN4965 Ccr4-Not transcription complex subunit
AN4650 Uncharacterized protein, conserved serine-proline rich region
The active and the inactive UspA pull 21 proteins related to fungal primary metabolism.
Furthermore, UspA associates with eight proteins classified to the nuclear transport category and additionally seven proteins related to transcriptional processing. This suggests a function of the deubiquitinase in nuclear transport of proteins by deubiquitination reactions. The cellular localization of the fusion protein mainly close to nuclei with subpopulations in the nucleus or in the cytoplasm corroborates this hypothesis (Figure 25). Furthermore, the karyopherins might regulate the subcellular localization of UspA itself. Thereby, UspA might function at the
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interphase of cytoplasmic and nuclear transport of proteins or mRNAs, which might be regulated through its deubiquitination acitvity. The specific task of UspA might thereby be dependent on its localization, which in turn could be determined through the karyopherins.
Table 16: Proteins exclusively identified in UspA-GFP pull down.
Description of identified proteins derive from information on AspGD or FungiDB (Cerqueira et al., 2014; Stajich et al., 2012). Domain predictions for uncharacterized proteins were performed with NCBI CD domain prediction tool (Marchler-Bauer et al., 2015).
Systematic
Name Description
Primary metabolism
AN2947 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase AN5883 Methylenetetrahydrofolate reductase, MetF
AN2493 Extracellular phytase, gluconate metabolism Transcriptional processing
AN03955 Uncharacterized protein, orthologs function in RNA metabolic process AN11128 RNA polymerase II transcription elongation factor, Ctr9
AN7480 Differentiation regulator, Nrd1, RNA binding AN4024 RNA maintenance of telomere capping protein 1
3.6 UspA ensures coordinated fungal development and secondary metabolism