Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome

Abstract

Dsk2p from Saccharomyces cerevisiae belongs to the class of proteins that contain a ubiquitin-like (UbL) domain at the N terminus together with a ubiquitin-associated (UBA) domain at the C terminus. We show here that the C-terminal UBA domain of Dsk2p binds to K48-linked polyubiquitin chains, and the N-terminal UbL domain of Dsk2p interacts with the proteasome. Overexpression of Dsk2p caused the accumulation of large amounts of polyubiquitin, and extragenic suppressors of the Dsk2p-mediated lethality proved to be temperature-sensitive mutations in two proteasome subunits, rpn1 and pre2. K48-linked ubiquitin-dependent degradation was impaired by disruption of the DSK2 gene. These results indicate that Dsk2p is K48-linked polyubiquitinbinding protein and also interacts with the proteasome. We discuss a possible role of adaptor function of Dsk2p via its UbL and UBA domains in the ubiquitin-proteasome pathway.ubiquitin-related protein ͉ polyubiquitin adaptor ͉ UBA domain ͉ ubiquitin chains ͉ UbL domain

Ubiquitin chains in the Dsk2 UBL domain mediate Dsk2 stability and protein degradation in yeast

“…Saccharomyces cerevisiae YPH499 (MATa ade2-101 leu2- trp1-63 ura3-52 lys2-801 his3-200), used as the wild-type strain, was previously described [17]. Mutant strains (dsk2 , rpn1-821, and pre2-75, dsk2  , dsk2 rad23 ), media, and growth conditions were described previously [17,21].…”

“…Mutant strains (dsk2 , rpn1-821, and pre2-75, dsk2  , dsk2 rad23 ), media, and growth conditions were described previously [17,21]. Plasmids carrying the glutathione S-transferase (GST)-tagged version pEG-KG-DSK2, were described previously [17].…”

“…Mutant strains (dsk2 , rpn1-821, and pre2-75, dsk2  , dsk2 rad23 ), media, and growth conditions were described previously [17,21]. Plasmids carrying the glutathione S-transferase (GST)-tagged version pEG-KG-DSK2, were described previously [17]. Multi-copy plasmids pGAL1-YEplac195 harboring DSK2 or pGAL1-YEplac112 harboring His 6 -T7-ubiquitin were used for the ubiquitination assay [17].…”

“…Plasmids carrying the glutathione S-transferase (GST)-tagged version pEG-KG-DSK2, were described previously [17]. Multi-copy plasmids pGAL1-YEplac195 harboring DSK2 or pGAL1-YEplac112 harboring His 6 -T7-ubiquitin were used for the ubiquitination assay [17]. A single-copy plasmid pRS316 harboring DSK2 and its endogenous promoter was used for complementation of dsk2 .…”

“…Budding yeast Dsk2 is a UBL-UBA protein that helps deliver polyubiquitinated proteins to the proteasome [17][18][19]. To better understand the molecular mechanisms of UBL-UBA proteins in the ubiquitin-proteasome pathway, we investigated the role of Dsk2 ubiquitination using lysine mutants of the Dsk2 UBL domain.…”