TY - JOUR
T1 - Analysis of a degron-containing reporter protein GFP-CL1 reveals a role for SUMO1 in cytosolic protein quality control
AU - Wang, Wei
AU - Lu, Jian
AU - Yang, Wei Chih
AU - Spear, Eric D.
AU - Michaelis, Susan
AU - Matunis, Michael J.
N1 - Funding Information:
This work was supported by NIGMS grant R01 GM060980 to M. J. M. and NIH grant R35 GM127073 to S. M. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
We thank all current and past Matunis lab members for their continuous support of this project, Dr Susan Michaelis and lab members for their collaboration, and NIGMS for funding this project.
Publisher Copyright:
© 2022 The Authors
PY - 2023/2
Y1 - 2023/2
N2 - Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential for maintaining proteostasis and normal cellular functions. Defects in PQC can result in disease, including cancer, cardiovascular disease, and neurodegeneration. The small ubiquitin-related modifiers (SUMOs) were previously implicated in the degradation of nuclear misfolded proteins, but their functions in cytoplasmic PQC are unclear. Here, in a systematic screen of SUMO protein mutations in the budding yeast Saccharomyces cerevisiae, we identified a mutant allele (Smt3-K38A/K40A) that sensitizes cells to proteotoxic stress induced by amino acid analogs. Smt3-K38A/K40A mutant strains also exhibited a defect in the turnover of a soluble PQC model substrate containing the CL1 degron (NES-GFP-Ura3-CL1) localized in the cytoplasm, but not the nucleus. Using human U2OS SUMO1- and SUMO2-KO cell lines, we observed a similar SUMO-dependent pathway for degradation of the mammalian degron-containing PQC reporter protein, GFP-CL1, also only in the cytoplasm but not the nucleus. Moreover, we found that turnover of GFP-CL1 in the cytoplasm was uniquely dependent on SUMO1 but not the SUMO2 paralogue. Additionally, we showed that turnover of GFP-CL1 in the cytoplasm is dependent on the AAA-ATPase, Cdc48/p97. Cellular fractionation studies and analysis of a SUMO1-GFP-CL1 fusion protein revealed that SUMO1 promotes cytoplasmic misfolded protein degradation by maintaining substrate solubility. Collectively, our findings reveal a conserved and previously unrecognized role for SUMO1 in regulating cytoplasmic PQC and provide valuable insights into the roles of sumoylation in PQC-associated diseases.
AB - Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential for maintaining proteostasis and normal cellular functions. Defects in PQC can result in disease, including cancer, cardiovascular disease, and neurodegeneration. The small ubiquitin-related modifiers (SUMOs) were previously implicated in the degradation of nuclear misfolded proteins, but their functions in cytoplasmic PQC are unclear. Here, in a systematic screen of SUMO protein mutations in the budding yeast Saccharomyces cerevisiae, we identified a mutant allele (Smt3-K38A/K40A) that sensitizes cells to proteotoxic stress induced by amino acid analogs. Smt3-K38A/K40A mutant strains also exhibited a defect in the turnover of a soluble PQC model substrate containing the CL1 degron (NES-GFP-Ura3-CL1) localized in the cytoplasm, but not the nucleus. Using human U2OS SUMO1- and SUMO2-KO cell lines, we observed a similar SUMO-dependent pathway for degradation of the mammalian degron-containing PQC reporter protein, GFP-CL1, also only in the cytoplasm but not the nucleus. Moreover, we found that turnover of GFP-CL1 in the cytoplasm was uniquely dependent on SUMO1 but not the SUMO2 paralogue. Additionally, we showed that turnover of GFP-CL1 in the cytoplasm is dependent on the AAA-ATPase, Cdc48/p97. Cellular fractionation studies and analysis of a SUMO1-GFP-CL1 fusion protein revealed that SUMO1 promotes cytoplasmic misfolded protein degradation by maintaining substrate solubility. Collectively, our findings reveal a conserved and previously unrecognized role for SUMO1 in regulating cytoplasmic PQC and provide valuable insights into the roles of sumoylation in PQC-associated diseases.
KW - CL1 degron
KW - Cdc48/p97
KW - protein quality control
KW - small ubiquitin-like modifier
KW - ubiquitin-proteasome system (UPS)
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U2 - 10.1016/j.jbc.2022.102851
DO - 10.1016/j.jbc.2022.102851
M3 - Article
C2 - 36587767
AN - SCOPUS:85146928618
SN - 0021-9258
VL - 299
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 2
M1 - 102851
ER -