Bul1, a New Protein That Binds to the Rsp5 Ubiquitin Ligase in Saccharomyces cerevisiae

Abstract

We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt- phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5. Furthermore, both Bul1 and Rsp5 appeared to be associated with large complexes which were separated through a sucrose gradient centrifugation, and Rsp5 was coimmunoprecipitated with Bul1. We discuss the possibility that Bul1 functions together with Rsp5 in protein ubiquitination.

Aneuploidy shortens replicative lifespan in Saccharomyces cerevisiae

“…Instead, damaged membrane proteins are targeted for endocytosis and pass through the multivesicular body (MVB) and to the lysosome (in yeast, the vacuole), where they are eventually degraded (Piper & Luzio, 2007; Zhao et al ., 2013). Bul1 plays an important role in this plasma membrane quality control system by facilitating the Rsp5 ‐ dependent polyubiquitination of multiple protein targets which directs their sorting to the vacuole for degradation (Yashiroda et al ., 1996; De Craene et al ., 2001; Helliwell et al ., 2001; Crespo et al ., 2004; Merhi & André, 2012; O'Donnell, 2012; Zhao et al ., 2013). Disruption of this membrane protein quality control pathway has been previously linked to accelerated aging (Fabrizio et al ., 2010), while the Rsp5/Bul1 node, in particular, has been shown to ameliorate the effects of protein aggregation in a model of neurodegenerative diseases (Tardiff et al ., 2013).…”

Human ubiquitin‐protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin‐conjugating enzymes

“…(i) Saccharomyces cerevisiae Rsp5 was originally identified as a revertant of mutations in the Spt3 gene encoding a TFIID-binding protein (Eisenmann et al 1992). Rsp5 is also required for the degradation of Gap1 and Fur4 permeases (Hein et al 1995), the endocytosis of uracil permease (Galan et al 1996), and the minichromosome maintenance (Yashiroda et al 1996). The large subunit of RNA polymerase II is a substrate of Rsp5 , and Schizosaccharomyces pombe Pub1 seems to be involved in the degradation of cdc25 phosphatase (Nefsky & Beach 1996).…”

Inositol Deacylation by Bst1p Is Required for the Quality Control of Glycosylphosphatidylinositol-anchored Proteins

“…We also confirmed that HA-Gas1*p was not affected in the different background of hrd1⌬ doa10⌬ double mutant cells (our unpublished data). We used the rsp5-101 mutant cells to test the involvement of Rsp5p in the Gas1*p degradation (Yashiroda et al, 1996). HA-Gas1*p was not stabilized in this mutant ( Figure 5B).…”