Abstract
Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting "receptors," the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Amino Acid Substitution
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Binding Sites
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Carrier Proteins / chemistry
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Carrier Proteins / metabolism*
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Cells, Cultured
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Conserved Sequence
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Endosomal Sorting Complexes Required for Transport
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Endosomes / metabolism*
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Fungal Proteins / chemistry
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Fungal Proteins / genetics
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Fungal Proteins / metabolism
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Lysosomes / chemistry
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Lysosomes / metabolism
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Models, Molecular
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Peptides / metabolism
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Protein Structure, Tertiary
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Protein Transport / physiology*
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Receptors, Cytoplasmic and Nuclear / metabolism*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Saccharomyces cerevisiae / cytology
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / metabolism*
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Sequence Deletion
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Sequence Homology, Amino Acid
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Transport Vesicles / metabolism
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Ubiquitin / chemistry
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Ubiquitin / genetics
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Ubiquitin / metabolism*
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Vesicular Transport Proteins*
Substances
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Carrier Proteins
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Endosomal Sorting Complexes Required for Transport
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Fungal Proteins
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Hse1 protein, S cerevisiae
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Peptides
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Receptors, Cytoplasmic and Nuclear
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Recombinant Fusion Proteins
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STP22 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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Ubiquitin
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VPS27 protein, S cerevisiae
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Vesicular Transport Proteins