The ubiquitin-proteasome system regulates membrane fusion of yeast vacuoles.
Ubiquitination is known to regulate early stages of intracellular vesicular transport, without proteasomal involvement. We now show that, in yeast, ubiquitination regulates a late-stage, membrane fusion, with proteasomal involvement. A known proteasome mutant had a vacuolar fragmentation phenotype in vivo often associated with vacuolar membrane fusion defects, suggesting a proteasomal role in fusion. Inhibiting vacuolar proteasomes interfered with membrane fusion in vitro, showing that fusion cannot occur without proteasomal degradation. If so, one would expect to find ubiquitinated proteins on vacuolar membranes. We found a small number of these, identified the most prevalent one as Ypt7 and mapped its two major ubiquitination sites. Ubiquitinated Ypt7 was linked to the degradation event that is necessary for fusion: vacuolar Ypt7 and vacuolar proteasomes were interdependent, ubiquitinated Ypt7 became a proteasomal substrate during fusion, and proteasome inhibitors reduced fusion to greater degree when we decreased Ypt7 ubiquitination. The strongest model holds that fusion cannot proceed without proteasomal degradation of ubiquitinated Ypt7. As Ypt7 is one of many Rab GTPases, ubiquitin-proteasome regulation may be involved in membrane fusion elsewhere.
Kleijnen
Maurits F
MF
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. maurits_kleijnen@hms.harvard.edu
Kirkpatrick
Donald S
DS
Gygi
Steven P
SP
D001665:Binding Sites; D008561:Membrane Fusion / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D030781:Organisms, Genetically Modified; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D061988:Proteasome Inhibitors; D011489:Protein Denaturation / Q000187:drug effects; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D025801:Ubiquitin / Q000502:physiology*; D014617:Vacuoles / Q000502:physiology*; D015003:Yeasts; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology
D008565:Membrane Proteins; D061988:Proteasome Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins