Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae.

Abstract

A library of random 10 residue peptides fused to the N‐terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N‐end rule pathway, a ubiquitin (Ub)‐dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N‐terminal residues. One of the N‐terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub‐dependent proteolytic system distinct from the N‐end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co‐immunoprecipitation and two‐hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub‐dependent proteolysis of test substrates. The discovery of the Ufd3p–Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.

Two Protein-tyrosine Phosphatases Inactivate the Osmotic Stress Response Pathway in Yeast by Targeting the Mitogen-activated Protein Kinase, Hog1

“…To test whether the GST-PTP fusions were functional in vivo, the pGST-PTP plasmids (see below) were transformed into TJY1, which bears the SLN1 deletion, sln1-⌬1::HIS3, and pSLN1-URA3. TJY1 is derived from JD51, a galactose-inducible strain (34). A strain bearing a disruption of the PTC1/ TPD1 gene was produced by transformation of DF5 with the tpd1::LEU2-3 allele, kindly provided by J. Broach (35).…”

Distinct Roles for the AAA ATPases NSF and p97 in the Secretory Pathway

“…These include roles in ubiquitin-and proteasome-dependent degradation of cytosolic proteins (Ghislain et al, 1996;Dai et al, 1998;Dai and Li, 2001), ER-associated degradation (ERAD; reviewed in Bays and Hampton, 2002;Tsai et al, 2002), and regulated ubiquitindependent processing (Rape et al, 2001 1997), Ufd1/Npl4 (Meyer et al, 2000(Meyer et al, , 2002, VCIP135 (Uchiyama et al, 2002), and SVIP (Nagahama et al, 2003). Although the mechanism by which AAA ATPases such as NSF and p97 use ATP to modulate the structure of their substrates is not known, the overall homology between these two enzymes makes the idea that they might operate by a similar mechanism attractive.…”

“…These include roles in ubiquitin-and proteasome-dependent degradation of cytosolic proteins (Ghislain et al, 1996;Dai et al, 1998;Dai and Li, 2001), ER-associated degradation (ERAD; reviewed in Bays and Hampton, 2002;Tsai et al, 2002), and regulated ubiquitindependent processing (Rape et al, 2001). It is believed that different adaptor proteins direct p97 to perform these varied cellular activities.…”

Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum

“…Recently, CDC48 has also been found to interact with Ufd3p, a WD40-repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae [17].…”