title: Perturbations to the ubiquitin conjugate proteome in yeast δubx mutants identify Ubx2 as a regulator of membrane lipid composition.
abstract: Yeast Cdc48 (p97/VCP in human cells) is a hexameric AAA ATPase that is thought to use ATP hydrolysis to power the segregation of ubiquitin-conjugated proteins from tightly bound partners. Current models posit that Cdc48 is linked to its substrates through adaptor proteins, including a family of seven proteins (13 in human) that contain a Cdc48-binding UBX domain. However, few substrates for specific UBX proteins are known, and hence the generality of this hypothesis remains untested. Here, we use mass spectrometry to identify ubiquitin conjugates that accumulate in cdc48 and ubx mutants. Different ubx mutants exhibit unique patterns of conjugate accumulation that point to functional specialization of individual Ubx proteins. To validate our findings, we examined in detail the endoplasmic reticulum-bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. Mutant ubx2Δ cells are deficient in both cleaving the ubiquitinated 120 kDa precursor of Spt23 to form active p90 and in localizing p90 to the nucleus, resulting in reduced expression of the target gene OLE1, which encodes fatty acid desaturase. Our findings provide a resource for future investigations on Cdc48, illustrate the utility of proteomics to identify ligands for specific ubiquitin receptor pathways, and uncover Ubx2 as a key player in the regulation of membrane lipid biosynthesis.
authors.affiliation: Division of Biology.
authors.forename: Natalie
authors.initials: N
authors.lastname: Kolawa
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.forename: Robert L J
authors.initials: RL
authors.lastname: Graham
authors.forename: Robert
authors.initials: R
authors.lastname: Oania
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D020543:Proteome; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D000074405:Valosin Containing Protein
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C542266:MGA2 protein, S cerevisiae; D008563:Membrane Lipids; D008565:Membrane Proteins; D020543:Proteome; C087570:SPT23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C507001:UBX2 protein, S cerevisiae; D025801:Ubiquitin; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; C000614178:VCP protein, human; D000074405:Valosin Containing Protein