Numerous nascent proteins undergo folding and maturation within the luminal and membrane compartments of the endoplasmic reticulum (ER). Despite the presence of various factors in the ER that promote protein folding, many proteins fail to properly fold and assemble and are subsequently degraded. Regulatory proteins in the ER also undergo degradation in a way that is responsive to stimuli or the changing needs of the cell. As in most cellular compartments, the ubiquitin-proteasome system (UPS) is responsible for the majority of the degradation at the ER—in a process termed ER-associated degradation (ERAD). Autophagic processes utilizing ubiquitin-like protein-conjugating systems also play roles in protein degradation at the ER. The ER is continuous with the nuclear envelope (NE), which consists of the outer nuclear membrane (ONM) and inner nuclear membrane (INM). While ERAD is known also to occur at the NE, only some of the ERAD ubiquitin-ligation pathways function at the INM. Protein degradation machineries in the ER/NE target a wide variety of substrates in multiple cellular compartments, including the cytoplasm, nucleoplasm, ER lumen, ER membrane, and the NE. Here, we review the protein degradation machineries of the ER and NE and the underlying mechanisms dictating recognition and processing of substrates by these machineries.

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The gene encoding Aspergillus nidulans acetamidase (amdS) was placed under control of Candida albicans ACT1 promoter and terminator sequences and then cloned into a plasmid containing C. glabrata ARS10,CEN8 or ARS10+CEN8 sequences. All plasmids transformed C. glabrata wild-type cells to acetamide+, with the ARS-only containing plasmid transforming cells at the highest frequencies (>1.0 × 10(4) transformants μg(-1)). Plasmids were rapidly lost under non-selective conditions with the frequency dependent on chromosomal element, thus recycling the acetamide- phenotype. The amdS plasmid was used to transform a set of clinical isolates resistant to a variety of antifungal drugs. All strains were successfully transformed to the acetamide+ phenotype at high frequency, confirming that this plasmid construct could be used as a simple dominant marker on virtually any strain. Gap repair experiments demonstrated that just as in Saccharomyces cerevisiae, gap repair functions efficiently inC. glabrata, suggesting that C. glabrata has numerous similarities toS. cerevisiae with regard to ease of molecular manipulation. The amdS system is inexpensive and efficient, and combined with existing C. glabrata plasmid elements, confers a high transformation frequency for C. glabrata with a phenotype that can be easily recycled.

Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.