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.

Prior to their delivery to and degradation by the 26S proteasome, misfolded transmembrane proteins of the ER and inner-nuclear membrane must be extracted from lipid bilayers. This extraction process, known as retrotranslocation, requires both quality-control E3 ubiquitin ligases and dislocation factors that diminish the energetic cost of dislodging the transmembrane segments of a protein. Recently, we showed that retrotranslocation of all ER transmembrane proteins requires the Dfm1 rhomboid pseudoprotease. However, we did not investigate whether Dfm1 also mediated retrotranslocation of transmembrane substrates in the inner-nuclear membrane (INM), which is contiguous with the ER but functionally separated from it by nucleoporins. Here, we show that canonical retrotranslocation occurs during INM-associated degradation (INMAD) but proceeds independently of Dfm1. Despite this independence, ERAD-M and INMAD cooperate to mitigate proteotoxicity. We show a novel misfolded-transmembrane-protein toxicity that elicits genetic suppression, demonstrating the cell's ability to tolerate a toxic burden of misfolded transmembrane proteins without functional INMAD or ERAD-M. This strikingly contrasted the suppression of the dfm1Δ null, which leads to the resumption of ERAD-M through HRD-complex remodeling. Thus, we conclude that INM retrotranslocation proceeds through a novel, private channel, which can be studied by virtue of its role in alleviating membrane-associated proteotoxicity.