Negative regulation of Candida glabrata Pdr1 by the deubiquitinase subunit Bre5 occurs in a ubiquitin independent manner

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Abstract

The primary route for development of azole resistance in the fungal pathogen Candida glabrata is acquisition of a point mutation in the PDR1 gene. This locus encodes a transcription factor that upon mutation drives high level expression of a range of genes including the ATP-binding cassette transporter-encoding gene CDR1. Pdr1 activity is also elevated in cells that lack the mitochondrial genome (ρ 0 cells), with associated high expression of CDR1 driving azole resistance. To gain insight into the mechanisms controlling activity of Pdr1, we expressed a tandem affinity purification (TAP)-tagged form of Pdr1 in both wild-type (ρ +) and ρ 0 cells. Purified proteins were analyzed by multidimensional protein identification technology (MudPIT) mass spectrometry identifying a protein called Bre5 as a factor that co-purified with TAP-Pdr1. In Saccharomyces cerevisiae, Bre5 is part of a deubiquitinase complex formed by association with the ubiquitin-specific protease Ubp3. Genetic analyses in C. glabrata revealed that loss of BRE5, but not UBP3, led to an increase in expression of PDR1 and CDR1 at the transcriptional level. These studies support the view that Bre5 acts as a negative regulator of Pdr1 transcriptional activity and behaves as a C. glabrata-specific modulator of azole resistance. <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/377c173f-8634-4965-a0b0-68fa77cdde86/PubMedCentral/image/nihms-1007185-f0001.jpg"/> </div> Bre5 is a known deubiquitinase subunit in Saccharomyces cerevisiae. We demonstrate that Bre5 is a negative regulator of Pdr1-dependent transcription in Candida glabrata, although this function is not conserved in S. cerevisiae. Induction of Pdr1 transcriptional activation is associated with decreased association of Bre5 with Pdr1 and we suggest that this represents a ubiquitin independent role for this protein.

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The tandem affinity purification (TAP) method: a general procedure of protein complex purification.

O Puig, F Caspary, G Rigaut … (2001)

Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration. Copyright 2001 Academic Press.

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Mechanism and function of deubiquitinating enzymes.

Alexander Y. Amerik, Mark Hochstrasser (2004)

Attachment of ubiquitin to proteins is a crucial step in many cellular regulatory mechanisms and contributes to numerous biological processes, including embryonic development, the cell cycle, growth control, and prevention of neurodegeneration. In these diverse regulatory settings, the most widespread mechanism of ubiquitin action is probably in the context of protein degradation. Polyubiquitin attachment targets many intracellular proteins for degradation by the proteasome, and (mono)ubiquitination is often required for down-regulating plasma membrane proteins by targeting them to the vacuole (lysosome). Ubiquitin-protein conjugates are highly dynamic structures. While an array of enzymes directs the conjugation of ubiquitin to substrates, there are also dozens of deubiquitinating enzymes (DUBs) that can reverse the process. Several lines of evidence indicate that DUBs are important regulators of the ubiquitin system. These enzymes are responsible for processing inactive ubiquitin precursors, proofreading ubiquitin-protein conjugates, removing ubiquitin from cellular adducts, and keeping the 26S proteasome free of inhibitory ubiquitin chains. The present review focuses on recent discoveries that have led to a better understanding the mechanisms and physiological roles of this diverse and still poorly understood group of enzymes. We also discuss briefly some of the proteases that act on ubiquitin-like protein (UBL) conjugates and compare them to DUBs.

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Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease.

Claudine Kraft, Anna Deplazes, Marc Sohrmann … (2008)

Eukaryotic cells use autophagy and the ubiquitin-proteasome system (UPS) as their major protein degradation pathways. Whereas the UPS is required for the rapid degradation of proteins when fast adaptation is needed, autophagy pathways selectively remove protein aggregates and damaged or excess organelles. However, little is known about the targets and mechanisms that provide specificity to this process. Here we show that mature ribosomes are rapidly degraded by autophagy upon nutrient starvation in Saccharomyces cerevisiae. Surprisingly, this degradation not only occurs by a non-selective mechanism, but also involves a novel type of selective autophagy, which we term 'ribophagy'. A genetic screen revealed that selective degradation of ribosomes requires catalytic activity of the Ubp3p/Bre5p ubiquitin protease. Although ubp3Delta and bre5Delta cells strongly accumulate 60S ribosomal particles upon starvation, they are proficient in starvation sensing and in general trafficking and autophagy pathways. Moreover, ubiquitination of several ribosomal subunits and/or ribosome-associated proteins was specifically enriched in ubp3Delta cells, suggesting that the regulation of ribophagy by ubiquitination may be direct. Interestingly, ubp3Delta cells are sensitive to rapamycin and nutrient starvation, implying that selective degradation of ribosomes is functionally important in vivo. Taken together, our results suggest a link between ubiquitination and the regulated degradation of mature ribosomes by autophagy.