Protein misfolding is monitored by a variety of cellular "quality control" systems. Endoplasmic reticulum (ER) quality control handles misfolded secretory and membrane proteins and is well characterized. However, less is known about the quality control of misfolded cytosolic proteins (CytoQC). To study CytoQC, we have employed a genetic system in Saccharomyces cerevisiae using a transplantable degron, CL1 (1). Attachment of CL1 to the cytosolic protein Ura3p destabilizes Ura3p, targeting it for rapid proteasomal degradation. We have performed a comprehensive analysis of Ura3p-CL1 degradation requirements. As shown previously, we observe that the ER-localized ubiquitin E2 (Ubc6p, Ubc7p, and Cue1p) and E3 (Doa10p) machinery involved in ER-associated degradation (ERAD) are also responsible for the degradation of the cytosolic substrate Ura3p-CL1. Importantly, we find that the cytosol/ER membrane-localized chaperones Ydj1p and Ssa1p, known to be necessary for the ERAD of membrane proteins with misfolded cytosolic domains, are also required for the ubiquitination and degradation of Ura3p-CL1. In addition, we show a role for the Cdc48p-Npl4p-Ufd1p complex in the degradation of Ura3p-CL1. When ubiquitination is blocked, a portion of Ura3p-CL1 is ER membrane-localized. Furthermore, access to the cytosolic face of the ER is required for the degradation of CL1 degroncontaining proteins. The ER is distributed throughout the cytosol, and our data, together with previous studies, suggest that the cytosolic face of the ER membrane serves as a "platform" for the degradation of Ura3p-CL1, which may also be the case for other CytoQC substrates.Mutation, errors in transcription or translation, and cellular stress can cause alterations in amino acids that may prevent proteins from attaining their properly folded, native conformations. Protein "quality control" is an essential process monitoring protein folding, ultimately targeting misfolded proteins for degradation via the ubiquitin-proteasome system. The importance of protein quality control is best exemplified by the numerous human diseases that can result from protein misfolding due to mutational or physiological causes and include cystic fibrosis, Parkinson disease, and ␣ 1 -antitrypsin deficiency (2, 3).Distinct protein quality control systems appear to exist in various cellular compartments, including the nucleus, mitochondria, and endoplasmic reticulum (ER), 2 with the bestcharacterized system being ER quality control (4 -7). Studies of ER quality control and, in particular, ER-associated degradation (ERAD) have revealed discrete chaperone and ubiquitination machinery required for the recognition and ubiquitination of different classes of misfolded secretory or membrane proteins. Much of this work has been greatly aided by the use of "model" ER quality control substrates, such as CPY* or Ste6p*, in the yeast Saccharomyces cerevisiae (8 -12). For example, it has become clear that model membrane proteins with misfolded cytosolic domains (called ERAD-C substrates), such as St...
Mitochondrial dysfunction is at the core of many diseases, ranging from inherited metabolic diseases to common conditions that are associated with ageing. While associations between ageing and mitochondrial function have been identified using mammalian models, much of the mechanistic insight has emerged from C. elegans. Mitochondrial respiration is recognized as an indicator of mitochondrial health. Seahorse XF96 respirometers are the state-of-the-art platform to assess respiration in cells, and we adapted the technique for applications involving C. elegans. Here, we provide a detailed protocol to optimise and measure respiration in C. elegans with the XF96 respirometer, including the interpretation of parameters and results. The protocol takes ~2 days to complete, excluding time spent culturing C. elegans, and includes (i) the preparation of C. elegans samples, (ii) selection and loading of compounds to be injected, (iii) preparing and executing a run with the XF96 respirometer, and (iv) post-experimental data-analysis, including normalization. In addition, we compare our XF96 application with other existing techniques, including the 8-well Seahorse XFp. The main benefits of the XF96 include the limited number of worms required and high-throughput capacity due to 96-well format.
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