Degradation of a cytosolic protein requires endoplasmic reticulum-associated degradation machinery.
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 degron-containing 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.
Metzger
Meredith Boyle
MB
Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Maurer
Matthew J
MJ
Dancy
Beverley M
BM
Michaelis
Susan
S
D000251:Adenosine Triphosphatases / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D012995:Solubility; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism
C109280:CUE1 protein, S cerevisiae; D002352:Carrier Proteins; D005656:Fungal Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; C070319:YDJ1 protein, S cerevisiae; C082461:UBC6 protein, S cerevisiae; C077072:UBC7 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae