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Perturbations to the Ubiquitin Conjugate Proteome in Yeast Δubx Mutants Identify Ubx2 as a Regulator of Membrane Lipid Composition*

Open AccessPublished:June 22, 2013DOI:https://doi.org/10.1074/mcp.M113.030163
      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.
      Budding yeast Cdc48 is an essential, highly abundant member of the AAA (ATPase associated with various cellular activities) protein family. Cdc48 has been linked to numerous functions throughout the cell but is best known for its critical role in ERAD (endoplasmic reticulum associated protein degradation)
      The abbreviations used are:
      ERAD
      endoplasmic reticulum associated protein degradation
      UPS
      ubiquitin protease system
      ER
      endoplasmic reticulum
      SFA
      saturated fatty acids
      UFA
      unsaturated fatty acids
      YPD
      yeast extract/peptone/dextrose.
      1The abbreviations used are:ERAD
      endoplasmic reticulum associated protein degradation
      UPS
      ubiquitin protease system
      ER
      endoplasmic reticulum
      SFA
      saturated fatty acids
      UFA
      unsaturated fatty acids
      YPD
      yeast extract/peptone/dextrose.
      , which occurs via the ubiquitin proteasome system (UPS). It is also involved in cell-cycle progression, homotypic membrane fusion, chromatin remodeling, autophagy, and transcriptional and metabolic regulation (
      • Stolz A.
      • Hilt W.
      • Buchberger A.
      • Wolf D.H.
      Cdc48: a power machine in protein degradation.
      ,
      • Meusser B.
      • Hirsch C.
      • Jarosch E.
      • Sommer T.
      ERAD: the long road to destruction.
      ,
      • Meyer H.
      • Bug M.
      • Bremer S.
      Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system.
      ,
      • Ye Y.
      Diverse functions with a common regulator: ubiquitin takes command of an AAA ATPase.
      ). Human Cdc48, known as p97 or VCP, has been the subject of much attention over the last few years because of its causal links to amyotrophic lateral sclerosis and inclusion body myopathy, Paget's disease of the bone, and frontotemporal dementia as well as its implied role in a variety of diseases including cancer (
      • Johnson J.O.
      • Mandrioli J.
      • Benatar M.
      • Abramzon Y.
      • Van Deerlin V.M.
      • Trojanowski J.Q.
      • Gibbs J.R.
      • Brunetti M.
      • Gronka S.
      • Wuu J.
      • Ding J.
      • McCluskey L.
      • Martinez-Lage M.
      • Falcone D.
      • Hernandez D.G.
      • Arepalli S.
      • Chong S.
      • Schymick J.C.
      • Rothstein J.
      • Landi F.
      • Wang Y.D.
      • Calvo A.
      • Mora G.
      • Sabatelli M.
      • Monsurrò M.R.
      • Battistini S.
      • Salvi F.
      • Spataro R.
      • Sola P.
      • Borghero G.
      • Galassi G.
      • Scholz S.W.
      • Taylor J.P.
      • Restagno G.
      • Chiò A.
      • Traynor B.J.
      Exome sequencing reveals VCP mutations as a cause of familial ALS.
      ,
      • Neumann M.
      • Kwong L.K.
      • Truax A.C.
      • Vanmassenhove B.
      • Kretzschmar H.A.
      • Van Deerlin V.M.
      • Clark C.M.
      • Grossman M.
      • Miller B.L.
      • Trojanowski J.Q.
      • Lee V.M.
      TDP-43-positive white matter pathology in frontotemporal lobar degeneration with ubiquitin-positive inclusions.
      ,
      • Chapman E.
      • Fry A.N.
      • Kang M.
      The complexities of p97 function in health and disease.
      ,
      • Haines D.S.
      p97-containing complexes in proliferation control and cancer: emerging culprits or guilt by association?.
      ,
      • Watts G.D.
      • Thomasova D.
      • Ramdeen S.K.
      • Fulchiero E.C.
      • Mehta S.G.
      • Drachman D.A.
      • Weihl C.C.
      • Jamrozik Z.
      • Kwiecinski H.
      • Kaminska A.
      • Kimonis V.E.
      Novel VCP mutations in inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia.
      ). As a result, p97 has been the target of multiple drug development efforts (
      • Chou T.F.
      • Deshaies R.J.
      Development of p97 AAA ATPase inhibitors.
      ,
      • Bursavich M.G.
      • Parker D.P.
      • Willardsen J.A.
      • Gao Z.H.
      • Davis T.
      • Ostanin K.
      • Robinson R.
      • Peterson A.
      • Cimbora D.M.
      • Zhu J.F.
      • Richards B.
      2-Anilino-4-aryl-1,3-thiazole inhibitors of valosin-containing protein (VCP or p97).
      ).
      Cdc48/p97 interacts with a large number of putative substrate adaptors and cofactors, including a family of proteins (seven in yeast, 13 in human cells) that contain a UBX domain (
      • Kloppsteck P.
      • Ewens C.A.
      • Förster A.
      • Zhang X.
      • Freemont P.S.
      Regulation of p97 in the ubiquitin-proteasome system by the UBX protein-family.
      ,
      • Schuberth C.
      • Buchberger A.
      UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97.
      ). The UBX domain binds to the N-terminal region of p97 and proteins bearing this domain have been suggested to serve as interchangeable adaptors that target Cdc48/p97 to specific substrates. Although the functions and mechanism of action of Cdc48/p97 remain poorly understood, it is generally presumed that it uses ATP hydrolysis to fuel the extraction of ubiquitinated proteins from multisubunit complexes or membranes as a prelude to their degradation by the proteasome. Cdc48/p97 may also remodel protein–protein and protein–nucleic acid complexes in a manner that is not coupled to either prior ubiquitination (
      • Wilcox A.J.
      • Laney J.D.
      A ubiquitin-selective AAA-ATPase mediates transcriptional switching by remodelling a repressor-promoter DNA complex.
      ) or subsequent degradation by the proteasome (
      • Böhm S.
      • Buchberger A.
      The budding yeast Cdc48(Shp1) complex promotes cell cycle progression by positive regulation of protein phosphatase 1 (Glc7).
      ,
      • Kirchner P.
      • Bug M.
      • Meyer H.
      Ubiquitination of the N-terminal Region of Caveolin-1 Regulates Endosomal Sorting by the VCP/p97 AAA-ATPase.
      ).
      Based on the abundance of Cdc48/p97 and the complexity of the network of adaptor proteins for which it serves as the hub, Cdc48/p97 has the potential to exert a profound influence on the UPS. However, the number of known substrates of Cdc48/p97 remains relatively small, and smaller still is the number of substrates that have been linked to a specific UBX domain protein. Indeed, only a handful of specific cases are known, and in half of them proteasomal degradation is not the final outcome (
      • Böhm S.
      • Buchberger A.
      The budding yeast Cdc48(Shp1) complex promotes cell cycle progression by positive regulation of protein phosphatase 1 (Glc7).
      ,
      • Kirchner P.
      • Bug M.
      • Meyer H.
      Ubiquitination of the N-terminal Region of Caveolin-1 Regulates Endosomal Sorting by the VCP/p97 AAA-ATPase.
      ,
      • Alexandru G.
      • Graumann J.
      • Smith G.T.
      • Kolawa N.J.
      • Fang R.
      • Deshaies R.J.
      UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.
      ,
      • Verma R.
      • Oania R.
      • Fang R.
      • Smith G.T.
      • Deshaies R.J.
      Cdc48/p97 mediates UV-dependent turnover of RNA Pol II.
      ,
      • Zhang L.
      • Zhou F.
      • Li Y.
      • Drabsch Y.
      • Zhang J.
      • van Dam H.
      • ten Dijke P.
      Fas-associated factor 1 is a scaffold protein that promotes beta-transducin repeat-containing protein (beta-TrCP)-mediated beta-catenin ubiquitination and degradation.
      ,
      • Barbin L.
      • Eisele F.
      • Santt O.
      • Wolf D.H.
      The Cdc48-Ufd1-Npl4 complex is central in ubiquitin-proteasome triggered catabolite degradation of fructose-1,6-bisphosphatase.
      ,
      • Lee J.J.
      • Park J.K.
      • Jeong J.
      • Jeon H.
      • Yoon J.B.
      • Kim E.E.
      • Lee K.J.
      Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD).
      ,
      • Lee J.J.
      • Kim Y.M.
      • Jeong J.
      • Bae D.S.
      • Lee K.J.
      Ubiquitin-associated (UBA) domain in human Fas associated factor 1 inhibits tumor formation by promoting Hsp70 degradation.
      ). To understand why there is such a profusion of UBX domain proteins in cells, it will be important to determine what these proteins do to their substrates, which will require knowing what their substrates are. Therefore, a major goal of this work was to enable future investigations into the function and regulation of UBX proteins by assembling a catalogue of candidate substrates/targets.
      A key paradigm that guides our current understanding of Cdc48 function emerged from studies on the processing of the transcription factors Spt23 and Mga2 (
      • Hoppe T.
      • Matuschewski K.
      • Rape M.
      • Schlenker S.
      • Ulrich H.D.
      • Jentsch S.
      Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.
      ,
      • Rape M.
      • Hoppe T.
      • Gorr I.
      • Kalocay M.
      • Richly H.
      • Jentsch S.
      Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone.
      ,
      • Hitchcock A.L.
      • Krebber H.
      • Frietze S.
      • Lin A.
      • Latterich M.
      • Silver P.A.
      The conserved npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation.
      ). These seminal studies revealed the mechanism by which cells control their ratio of saturated to unsaturated fatty acids to maintain an appropriate lipid composition in cellular membranes. Central to this regulation are the transcription factors Spt23 and Mga2, which are released from the endoplasmic reticulum (ER) membrane and translocate to the nucleus to activate expression of OLE1, which encodes the stearoyl-Δ9 desaturase that governs the conversion of saturated fatty acids (SFAs) to unsaturated fatty acids (UFAs) (
      • Martin C.E.
      • Oh C.S.
      • Jiang Y.
      Regulation of long chain unsaturated fatty acid synthesis in yeast.
      ,
      • Zhang S.
      • Skalsky Y.
      • Garfinkel D.J.
      MGA2 or SPT23 is required for transcription of the delta9 fatty acid desaturase gene, OLE1, and nuclear membrane integrity in Saccharomyces cerevisiae.
      ). Spt23 and Mga2 are initially produced as 120 kDa precursors (p120) embedded in the ER by C-terminal signal or anchor domains, such that the bulk of each protein projects into the cytosol. The p120 form of each protein is ubiquitinated by Rsp5 and cleaved by the proteasome, which yields a p90 form that lacks the transmembrane signal or anchor (
      • Hoppe T.
      • Matuschewski K.
      • Rape M.
      • Schlenker S.
      • Ulrich H.D.
      • Jentsch S.
      Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.
      ,
      • Hitchcock A.L.
      • Krebber H.
      • Frietze S.
      • Lin A.
      • Latterich M.
      • Silver P.A.
      The conserved npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation.
      ,
      • Shcherbik N.
      • Zoladek T.
      • Nickels J.T.
      • Haines D.S.
      Rsp5p is required for ER bound Mga2p120 polyubiquitination and release of the processed/tethered transactivator Mga2p90.
      ,
      • Piwko W.
      • Jentsch S.
      Proteasome-mediated protein processing by bidirectional degradation initiated from an internal site.
      ,
      • Shcherbik N.
      • Kee Y.
      • Lyon N.
      • Huibregtse J.M.
      • Haines D.S.
      A single PXY motif located within the carboxyl terminus of Spt23p and Mga2p mediates a physical and functional interaction with ubiquitin ligase Rsp5p.
      ). However, p90 remains tethered to the ER through dimerization to an uncleaved p120, until it is disengaged from its partner by the “segregase” activity of Cdc48, acting in concert with Ufd1-Npl4 (
      • Rape M.
      • Hoppe T.
      • Gorr I.
      • Kalocay M.
      • Richly H.
      • Jentsch S.
      Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone.
      ,
      • Shcherbik N.
      • Haines D.S.
      Cdc48p(Npl4p/Ufd1p) binds and segregates membrane-anchored/tethered complexes via a polyubiquitin signal present on the anchors.
      ,
      • Braun S.
      • Matuschewski K.
      • Rape M.
      • Thoms S.
      • Jentsch S.
      Role of the ubiquitin-selective CDC48(UFD1/NPL4)chaperone (segregase) in ERAD of OLE1 and other substrates.
      ). The released p90 can then travel to the nucleus, where it activates expression of OLE1 (
      • Rape M.
      • Hoppe T.
      • Gorr I.
      • Kalocay M.
      • Richly H.
      • Jentsch S.
      Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone.
      ). The p90 species are quite unstable, and Cdc48 also promotes their degradation (
      • Rumpf S.
      • Jentsch S.
      Functional division of substrate processing cofactors of the ubiquitin-selective Cdc48 chaperone.
      ). In addition to acting as a transcription factor, Mga2 also influences the stability of OLE1 mRNA (
      • Kandasamy P.
      • Vemula M.
      • Oh C.S.
      • Chellappa R.
      • Martin C.E.
      Regulation of unsaturated fatty acid biosynthesis in Saccharomyces: the -endoplasmic reticulum membrane protein, Mga2p, a transcription activator of the OLE1 gene, regulates the stability of the OLE1 mRNA through exosome-mediated mechanisms.
      ), although the degree to which this function is carried out by p90 or p120 is not understood.

      Acknowledgments

      We thank S. Jentsch, L. Hicke, T. Sommer, E. Jarosch, and C.-W. Wang for gifts of strains, plasmids, antibodies, and W. den Besten for help with strain construction and valuable discussions. We also thank R. Ernst for communicating results before publication and M. Mann for hosting N.K. for a visit.

      Supplementary Material

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