Ssz1 Restores Endoplasmic Reticulum-Associated Protein Degradation in Cells Expressing Defective Cdc48–Ufd1–Npl4 Complex by Upregulating Cdc48

Abstract

The endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway eliminates aberrant proteins from the ER. The key role of Cdc48p-Ufd1p-Npl4p is indicated by impaired ERAD in Saccharomyces cerevisiae with mutations in any of this complex's genes. We identified SSZ1 in genetic screens for cdc48-10 suppressors and show that it upregulates Cdc48p via the pleiotropic drug resistance (PDR) network. A pSSZ1 plasmid restored impaired ERAD-M of 6myc-Hmg2 in cdc48-10, ufd1-2, and npl4-1, while SSZ1 deletion had no effect. Ssz1p activates Pdr1p, the PDR master regulator. Indeed, plasmids of PDR1 or its target gene RPN4 increased cdc48-10p levels and restored ERAD-M in cdc48-10. Rpn4p regulates transcription of proteasome subunits and CDC48, thus RPN4 deletion abolished ERAD. However, the diminished proteasome level in Drpn4 was sufficient for degrading a cytosolic substrate, whereas the impaired ERAD-M was the result of diminished Cdc48p and was restored by expression of pCDC48. The corrected ERAD-M in the hypomorphic strains of the Cdc48 partners ufd1-2 and npl4-1 by the pCDC48 plasmid, and in cdc48-10 cells by the pcdc48-10 plasmid, combined with the finding that neither pSSZ1 nor pcdc48-10 restored ERAD-L of CPY*-HA, support our conclusion that Ssz1p suppressing effects is brought about by upregulating Cdc48p.

The proteasome biogenesis regulator Rpn4 cooperates with the unfolded protein response to promote resistance to endoplasmic reticulum stress

“…Indeed, HAC1 splicing was elevated in untreated ∆rpn4 cells, consistent with high throughput data ( Figure 3C; Jonikas et al, 2009). The activation of the UPR in the absence of Rpn4 likely arises from inefficient ERAD and a subsequent buildup of misfolded proteins in the ER (Ng et al, 2000;Bosis et al, 2010). Accordingly, constitutive UPR signaling in ∆rpn4 cells was similar to that in cells lacking the ubiquitin ligase Hrd1, which is essential for ERAD.…”

“…Indeed, Yap1 not only activates RPN4 but is itself activated by Rpn4 (Mannhaupt et al, 1999) and may aid stress resistance by preventing oxidative damage. Furthermore, we directly tested the Rpn4 target gene CDC48 (Bosis et al, 2010), even though it was not found in the screen. Overexpression of CDC48 also restored growth of ∆hac1 cells expressing ngCPY*, although weakly compared to overexpression of RPN4 (Figures 2B -E).…”

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

“…The Zuo1-binding and nucleotide-binding domains could be eliminated individually, but Ssz1 is nonfunctional if both domains are disrupted in cis, suggesting that at least one intact domain is necessary for function. Independent of its roles in the ribosome, Ssz1 has been shown to activate Pdr1, a transcription factor associated with the induction of genes involved in the efflux of cytotoxic compounds, the stress response, lipid metabolism, and the ERassociated degradation (ERAD) pathway (33). Ssb is thought to be involved primarily in cotranslational protein folding and the promotion of nascent-chain movement through the exit tunnel.…”

Protein folding and quality control in the endoplasmic reticulum: Recent lessons from yeast and mammalian cell systems

“…In fact, genetic evidence indicates that ERAD-M substrates are most sensitive to Cdc48 depletion [115], and p97 dependence correlates with the relative stability of a polytopic membrane protein in the bilayer [116]. In contrast, some ERAD substrates are retrotranslocated in a Cdc48/p97-independent fashion and instead use an analogous AAA complex in the proteasome cap [117-120].…”