DOA1/UFD3 plays a role in sorting ubiquitinated membrane proteins into multivesicular bodies.
Ubiquitin (Ub) is a sorting signal that targets integral membrane proteins to the interior of the vacuole/lysosome by directing them into lumenal vesicles of multivesicular bodies (MVBs). The Vps27-Hse1 complex, which is homologous to the Hrs-STAM complex in mammalian cells, serves as a Ub-sorting receptor at the surface of early endosomes. We have found that Hse1 interacts with Doa1/Ufd3. Doa1 is known to interact with Cdc48/p97 and Ub and is required for maintaining Ub levels. We find that the Hse1 Src homology 3 domain binds directly to the central PFU domain of Doa1. Mutations in Doa1 that block Hse1 binding but not Ub binding do not alter Ub levels but do result in the missorting of the MVB cargo GFP-Cps1. Loss of Doa1 also causes a synthetic growth defect when combined with loss of Vps27. Unlike the loss of Doa1 alone, the doa1Delta vps27Delta double mutant phenotype is not suppressed by Ub overexpression, demonstrating that the effect is not due to indirect consequence of lowered Ub levels. Loss of Doa1 results in a defect in the accumulation of GFP-Ub within yeast vacuoles, implying that there is a reduction in the flux of ubiquitinated membrane proteins through the MVB pathway. This defect was also reflected by an inability to properly sort Vph1-GFP-Ub, a modified subunit of the multiprotein vacuolar ATPase complex, which carries an in-frame fusion of Ub as an MVB sorting signal. These results reveal novel roles for Doa1 in helping to process ubiquitinated membrane proteins for sorting into MVBs.
Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.
Jihui
J
Ren
Natasha
N
Pashkova
Stanley
S
Winistorfer
Robert C
RC
Piper
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D011992:Endosomes / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D012685:Sepharose / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C099944:DOA1 protein, S cerevisiae; D000939:Epitopes; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D012685:Sepharose; D005978:Glutathione