DOA1/UFD3 Plays a Role in Sorting Ubiquitinated Membrane Proteins into Multivesicular Bodies

Abstract

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 Ubsorting 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 doa1⌬ vps27⌬ 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 inframe 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.

Cargo ubiquitination is essential for multivesicular body intralumenal vesicle formation

“…Our previous studies showed that when Hse1-DUb is expressed in wild-type cells, either as the sole copy of Hse1 or in addition to endogenous Hse1, it effectively removes Ub from cargo during the MVB-sorting process, and blocks the sorting of a variety of cargoes that would normally undergo Ub-dependent sorting into MVB ILVs [7]. As a further measure of the effectiveness of Hse1-DUb to block MVB sorting of ubiquitinated cargo, we followed green fluorescent protein (GFP)-tagged Ub (GFP-Ub; [8]). Earlier studies indicated that GFP-Ub could be used as a proxy to monitor the delivery of a wide variety of cargoes into the MVB pathway.…”

“…Earlier studies indicated that GFP-Ub could be used as a proxy to monitor the delivery of a wide variety of cargoes into the MVB pathway. For instance, wild-type cells accumulate GFP-Ub in the vacuole and loss of the late-acting Doa4 DUb, which is required for removing much of the Ub from MVB cargo before its entry into ILVs, causes hyper-accumulation of GFP-Ub within the vacuole [8]. As expected, we find that GFP-Ub effectively enters into the general pool of Ub as it is found readily conjugated to a variety of proteins (Fig 1B), and it also accumulates in a processed form within the vacuole lumen in Pep þ cells ( Supplementary Fig S1 online).…”

Class E compartments form in response to ESCRT dysfunction in yeast due to hyperactivity of the Vps21 Rab GTPase

“…7) suggests how challenging it is to form these structures in the context of ESCRT function. The aberrant accumulation of ubiquitin at endosomes, which occurs in response to ESCRT dysfunction (Ren et al, 2008), might also be involved in class E compartment biogenesis because yeast lacking endosomal deubiquitylation activity do not form class E compartments unless cellular ubiquitin levels are maintained (Richter et al, 2007). Evidence that ubiquitylated cargoes inhibit endosomal maturation is the enhanced class E compartment morphology seen upon stimulation of epidermal growth factor receptor endocytosis after ESCRT-I depletion in human cells ( Razi and Futter, 2006).…”

Cellular Functions of Ufd2 and Ufd3 in Proteasomal Protein Degradation Depend on Cdc48 Binding

“…⌬ufd3 strains exhibit a number of stress phenotypes and negative genetic interactions, many of which are an indirect consequence of ubiquitin depletion (32,39,44,47,56). However, defects of ⌬ufd3 in the monoubiquitylation of histone H2B (39), in the sorting of ubiquitylated membrane proteins into multivesicular bodies for vacuolar degradation (54), and in ribophagy (48) were shown to persist upon ubiquitin overexpression, suggesting that Ufd3 is directly involved in these processes.…”