Abstract
Proteasome is a protein degradation complex that plays a major role in maintaining cellular homeostasis. Despite extensive efforts to identify protein substrates that are degraded through ubiquitination, the regulation of proteasome activity itself under diverse signals is poorly understood. In this study, we have isolated iRhom1 as a stimulator of proteasome activity from genome-wide functional screening using cDNA expression and an unstable GFP-degron. Downregulation of iRhom1 reduced enzymatic activity of proteasome complexes and overexpression of iRhom1 enhanced it. Native-gel and fractionation analyses revealed that knockdown of iRhom1 expression impaired the assembly of the proteasome complexes. The expression of iRhom1 was increased by endoplasmic reticulum (ER) stressors, such as thapsigargin and tunicamycin, leading to the enhancement of proteasome activity, especially in ER-containing microsomes. iRhom1 interacted with the 20S proteasome assembly chaperones PAC1 and PAC2, affecting their protein stability. Moreover, knockdown of iRhom1 expression impaired the dimerization of PAC1 and PAC2 under ER stress. In addition, iRhom1 deficiency in D. melanogaster accelerated the rough-eye phenotype of mutant Huntingtin, while transgenic flies expressing either human iRhom1 or Drosophila iRhom showed rescue of the rough-eye phenotype. Together, these results identify a novel regulator of proteasome activity, iRhom1, which functions via PAC1/2 under ER stress.
Highlights
After a gain-of-function screen using 6,200 cDNAs in mammalian expression vectors, we found several clones that greatly reduced the signal of GFPU upon overexpression
When we examined overexpression effects of more than ~100 cDNAs encoding endoplasmic reticulum (ER) membrane proteins, including amyloid precursor protein (APP), prolyl 4-hydroxylase subunit alpha-2(P4HA2), transmembrane emp[24] protein transport domain containing 5 (CGI-100), and glucose-6-phosphatase (G6PT), on proteasome activity, we could not observe any significant change in our assay employing GFPU (Figure S2a and b), indicating that elevation of proteasome activity by iRhom[1] is not artificial result of overexpressing a polytopic membrane protein
Because iRhom[1] is a member of the Rhomboid protease family that regulates the EGF quality control system in the ER16, we evaluated whether other members of the Rhomboid family affect proteasome activity
Summary
These previous findings suggest that the proteasome is regulated in a signaland tissue-specific manner with physiologic and pathologic relevance. The iRhom[1] and 2 are counter parts of drosophila iRhom, member of the Rhomboid protease family that is located in the ER and functions to process EGF or TGF-α. In contrast to other Rhomboid protease family members, iRhom lacks protease catalytic activity and acts as a pseudoprotease that inhibits translocation of EGF ligand family members to the Golgi by binding to them and targeting them to the proteasome. IRhom[1] and 2 participate promoting the degradation of EGF16. The expression of iRhom[1] was increased under ER stress and enhanced proteasome activity, possibly via PAC1 and PAC2
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