UspA activity requires C469 and C1066 to reduce the cellular pool of ubiquitinated proteins

The upregulation of the uspA transcript in the absence of a functional CSN complex as well as the association to certain CSN subunits to UspA in Y2H and BiFC experiments indicate a role of UspA in the ubiquitin-proteasome system. Deletion strains of uspA were constructed to elucidate the function of the DUB. The A. nidulans uspA ORF was replaced by the pyroA marker from Aspergillus fumigatus resulting in the ΔuspA^pyroA deletion strain. This strain was complemented by ectopical integration of the uspA ORF resulting in the comp^pyroA strain. Furthermore, an uspA deletion strain was constructed using a recyclable marker system in which the marker cassette can be excised off the genome and only a small six site is left as a scar. This strain was called ΔuspA^Six. The resulting strain was complemented by integration of the uspA::gfp fusion construct in the uspA gene locus through homologous recombination (uspA::gfp). Additionally, an UspA-GFP mutant protein was constructed, which carries amino acid exchanges: a cysteine residue belonging to the catalytic triad was mutated to alanine (C469A) and one cysteine residue of the zinc finger motif was mutated to alanine (C1066A). In the following this strain is referred to as uspA^AA::gfp.

qRT-PCR and western hybridization experiments were performed to analyze wether the point mutations in uspA^AA::gfp affect uspA gene transcription or translation (Figure 27). Mycelia of wild type strain, uspA::gfp or uspA^AA::gfp expressing strains were harvested after 20 h of growth in liquid cultures. After preparation of RNA and cDNA synthesis, qRT-PCRs were performed (Figure 27A). uspA::gfp and uspA^AA::gfp reveal two to three fold higher expression of the uspA gene compared to wild type expression levels (Figure 27A). Furthermore, protein levels of the fusion proteins were analyzed with western hybridization experiments (Figure 27B).

Quantification of the pixel density of the fusion protein band (red arrow) against the Ponceau S loading control revealed no significantly different fusion protein amounts.

Cellular protein ubiquitination levels during fungal development were analyzed in A. nidulans wild type, uspA deletion and complementation strains as well as in the strain expressing uspA^AA::gfp (Figure 28). Therefore, proteins were isolated after 20 h of vegetative growth in submerged culture, 24 h growth on solid agar plates in light to induce asexual development or 48 h of growth on agar plates in darkness with limited oxygen supply to induce sexual development. The amount of proteins modified with ubiquitin was analyzed by western hybridization using a αUbiquitin antibody. Deletion of uspA results in increased amounts of ubiquitinated proteins compared to the wild type at all tested growth states (Figure 28).

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Figure 27: The fusion protein UspA^AA-GFP is transcribed and expressed like UspA-GFP.

A) Mycelia derived from 20 h vegetatively grown cultures were used for RNA extraction and subsequent cDNA synthesis. Wild type expression level of uspA was set to 1. uspA::gfp and uspA^AA::gfp reveal two to three fold elevated expression than the wild type. h2A and 15S rRNA served as housekeeping genes. Results derive from two independent biological replicates with three technical replicates each. Error bars represent the standard error of the mean (SEM).

B) Western hybridization experiments of total protein crude extracts derived from vegetatively grown cultures were performed. Wild type crude extract was included as control to estimate the unspecific binding of the αGFP antibody. Signals of the fusion protein band (highlighted with red arrow) were quantified using Ponceau S staining as loading control. Error bars represent the SEM of two biological replicates.

This effect was independent from the utilized marker cassette. Ectopic integration of the uspA ORF into the genome of the deletion strain (comp^pyroA) as well as in locus complementation (uspA::gfp) restored the amount of ubiquitinated proteins to wild type level. The strain expressing UspA^AA-GFP shows an increase in the total amount of ubiquitinated proteins as well. The amount of modified proteins in the strain expressing the inactive UspA-GFP mutant is similar to both deletion strains. This indicates that the two point mutations render the UspA deubiquitinating enzyme inactive. The highest accumulation of ubiquitinated proteins was detected during asexual development in uspA deletion or inactive mutant strains. The level of proteins modified with ubiquitin is in these strains more than 2.5 times higher compared to the wild type and complementation strains.

This shows that UspA is a major deubiquitinase of A. nidulans, which is active throughout the whole fungal development. The two cysteine residues, one is part of the catalytic triad and the

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other one is a member of the zinc finger motif, seem to be essential for the deubiquitination activity. UspA-GFP without these amino acid substitutions showed wild type like levels of ubiquitinated proteins.

Figure 28: UspA deubiquitinates proteins during fungal growth and development.

Western hybridization of total protein crude extracts derived from mycelia grown for 20 h vegetatively in submerged cultures. For asexual and sexual development, mycelia were shifted from liquid cultures on solid agar plates and incubated for 24 h in light or darkness to induce respective development. Nitrocellulose membranes were incubated with αUbiquitin antibody.

Signals were normalized to the loading control (Ponceau S). Error bars represent the standard error of the mean (SEM) of at least two biological repetitions. Wild type ubiquitination levels were set to 1. The dashed lines indicate that the samples are not on the same membrane. The respective control strains used for quantification were loaded on all membranes.

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3.5 UspA interacts with proteins involved in nuclear transport, RNA processing and the