In Vivo Ubiquitin Linkage-type Analysis Reveals that the Cdc48-Rad23/Dsk2 Axis Contributes to K48-Linked Chain Specificity of the Proteasome

Hikaru Tsuchiya; Fumiaki Ohtake; Naoko Arai; Ai Kaiho; Sayaka Yasuda; Keiji Tanaka; Yasushi Saeki

doi:10.1016/j.molcel.2017.04.024

In Vivo Ubiquitin Linkage-type Analysis Reveals that the Cdc48-Rad23/Dsk2 Axis Contributes to K48-Linked Chain Specificity of the Proteasome

Mol Cell. 2017 May 18;66(4):488-502.e7. doi: 10.1016/j.molcel.2017.04.024.

Authors

Hikaru Tsuchiya¹, Fumiaki Ohtake¹, Naoko Arai¹, Ai Kaiho¹, Sayaka Yasuda¹, Keiji Tanaka², Yasushi Saeki³

Affiliations

¹ Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo 156-8506, Japan.
² Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo 156-8506, Japan. Electronic address: [email protected].
³ Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo 156-8506, Japan. Electronic address: [email protected].

PMID: 28525741
DOI: 10.1016/j.molcel.2017.04.024

Abstract

Ubiquitin-binding domain (UBD) proteins regulate numerous cellular processes, but their specificities toward ubiquitin chain types in cells remain obscure. Here, we perform a quantitative proteomic analysis of ubiquitin linkage-type selectivity of 14 UBD proteins and the proteasome in yeast. We find that K48-linked chains are directed to proteasomal degradation through selectivity of the Cdc48 cofactor Npl4. Mutating Cdc48 results in decreased selectivity, and lacking Rad23/Dsk2 abolishes interactions between ubiquitylated substrates and the proteasome. Among them, only Npl4 has K48 chain specificity in vitro. Thus, the Cdc48 complex functions as a K48 linkage-specifying factor upstream of Rad23/Dsk2 for proteasomal degradation. On the other hand, K63 chains are utilized in endocytic pathways, whereas both K48 and K63 chains are found in the MVB and autophagic pathways. Collectively, our results provide an overall picture of the ubiquitin network via UBD proteins and identify the Cdc48-Rad23/Dsk2 axis as a major route to the proteasome.

Keywords: MVB sorting; UBD protein; autophagy; endocytosis; mass spectrometry; proteasome; proteasome inhibitor; ubiquitin; ubiquitin-binding domain.

MeSH terms

Adenosine Triphosphatases / genetics
Adenosine Triphosphatases / metabolism*
Carrier Proteins / genetics
Carrier Proteins / metabolism
Cell Cycle Proteins / genetics
Cell Cycle Proteins / metabolism*
DNA-Binding Proteins / genetics
DNA-Binding Proteins / metabolism*
Endocytosis
Endosomal Sorting Complexes Required for Transport / genetics
Endosomal Sorting Complexes Required for Transport / metabolism
Membrane Proteins / genetics
Membrane Proteins / metabolism
Nucleocytoplasmic Transport Proteins / genetics
Nucleocytoplasmic Transport Proteins / metabolism
Proteasome Endopeptidase Complex / drug effects
Proteasome Endopeptidase Complex / genetics
Proteasome Endopeptidase Complex / metabolism*
Proteasome Inhibitors / pharmacology
Protein Binding
Protein Interaction Domains and Motifs
Proteolysis
Saccharomyces cerevisiae / drug effects
Saccharomyces cerevisiae / enzymology*
Saccharomyces cerevisiae / genetics
Saccharomyces cerevisiae Proteins / genetics
Saccharomyces cerevisiae Proteins / metabolism*
Time Factors
Ubiquitin / metabolism*
Ubiquitination* / drug effects
Ubiquitins / genetics
Ubiquitins / metabolism*
Valosin Containing Protein

Substances

CUE1 protein, S cerevisiae
Carrier Proteins
Cell Cycle Proteins
DNA-Binding Proteins
DSK2 protein, S cerevisiae
Endosomal Sorting Complexes Required for Transport
Membrane Proteins
NPL4 protein, S cerevisiae
Nucleocytoplasmic Transport Proteins
Proteasome Inhibitors
RAD23 protein, S cerevisiae
Saccharomyces cerevisiae Proteins
Ubiquitin
Ubiquitins
Proteasome Endopeptidase Complex
Adenosine Triphosphatases
CDC48 protein, S cerevisiae
Valosin Containing Protein