ABSTRACT
In both health and disease, the ubiquitin-proteasome system (UPS) degrades point mutants that retain partial function but have decreased stability compared with their wild-type counterparts. This class of UPS substrate includes routine translational errors and numerous human disease alleles, such as the most common cause of cystic fibrosis, ΔF508-CFTR. Yet, there is no systematic way to discover novel examples of these "minimally misfolded" substrates. To address that shortcoming, we designed a genetic screen to isolate functional-but-degraded point mutants, and we used the screen to study soluble, monomeric proteins with known structures. These simple parent proteins yielded diverse substrates, allowing us to investigate the structural features, cytotoxicity, and small-molecule regulation of minimal misfolding. Our screen can support numerous lines of inquiry, and it provides broad access to a class of poorly understood but biomedically critical quality-control substrates.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Humans , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Ubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational , Mutation/geneticsABSTRACT
Yeast recombination cloning is a straightforward and powerful method for recombining a plasmid backbone with a specific DNA fragment. However, the utility of yeast recombination cloning is limited by the requirement for the backbone to contain an CEN/ARS element, which allows for the recombined plasmids to propagate. Although yeast CEN/ARS plasmids are often suitable for further studies, we demonstrate here that they can vary considerably in copy number from cell to cell and from colony to colony. Variation in plasmid copy number can pose an unacceptable and often unacknowledged source of phenotypic variation. If expression levels are critical to experimentation, then constructs generated with yeast recombination cloning must be subcloned into integrating plasmids, a step that often abrogates the utility of recombination cloning. Accordingly, we have designed a vector that can be used for yeast recombination cloning but can be converted into the integrating version of the resulting vector without an additional subcloning. We call these "ICE" vectors, for "Integrating after CEN Excision." The ICE series was created by introducing a "rare-cutter" NotI-flanked CEN/ARS element into the multiple cloning sites of the pRS series yeast integration plasmids. Upon recovery from yeast, the CEN/ARS is excised by NotI digest and subsequently religated without need for purification or transfer to new conditions. Excision by this approach takes ~3 hr, allowing this refinement in the same time frame as standard recombination cloning.
