Ub-ProT reveals global length and composition of protein ubiquitylation in cells

Hikaru Tsuchiya; Daocharad Burana; Fumiaki Ohtake; Naoko Arai; Ai Kaiho; Masayuki Komada; Keiji Tanaka; Yasushi Saeki

doi:10.1038/s41467-018-02869-x

Ub-ProT reveals global length and composition of protein ubiquitylation in cells

Nat Commun. 2018 Feb 6;9(1):524. doi: 10.1038/s41467-018-02869-x.

Authors

Hikaru Tsuchiya¹, Daocharad Burana^{1

2}, Fumiaki Ohtake¹, Naoko Arai¹, Ai Kaiho¹, Masayuki Komada², Keiji Tanaka³, Yasushi Saeki⁴

Affiliations

¹ Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan.
² Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, 4259-B16 Nagatsuta, Midori, Yokohama, 226-8501, Japan.
³ Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan. [email protected].
⁴ Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan. [email protected].

Abstract

Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor receptor (EGFR) is rapidly modified with K63-linked tetra- to hexa-ubiquitin chains following EGF treatment in human cells. Thus, the Ub-ProT method can contribute to our understanding of mechanisms regulating physiological ubiquitin chain lengths and composition.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adaptor Proteins, Signal Transducing
Autophagy-Related Proteins
Carrier Proteins / genetics
Carrier Proteins / metabolism
Cell Cycle Proteins / genetics
Cell Cycle Proteins / metabolism
Epidermal Growth Factor / pharmacology
ErbB Receptors / metabolism*
HeLa Cells
Humans
Leupeptins / pharmacology
Polyubiquitin / analysis*
Polyubiquitin / chemistry
Polyubiquitin / metabolism
Proteins / metabolism*
Saccharomyces cerevisiae / drug effects
Saccharomyces cerevisiae / metabolism
Saccharomyces cerevisiae Proteins / genetics
Saccharomyces cerevisiae Proteins / metabolism*
Ubiquitin / chemistry
Ubiquitin / metabolism
Ubiquitinated Proteins / metabolism
Ubiquitination*
Valosin Containing Protein / genetics
Valosin Containing Protein / metabolism*

Substances

Adaptor Proteins, Signal Transducing
Autophagy-Related Proteins
Carrier Proteins
Cell Cycle Proteins
Leupeptins
Proteins
Saccharomyces cerevisiae Proteins
UBQLN1 protein, human
Ubiquitin
Ubiquitinated Proteins
Polyubiquitin
Epidermal Growth Factor
EGFR protein, human
ErbB Receptors
CDC48 protein, S cerevisiae
Valosin Containing Protein
benzyloxycarbonylleucyl-leucyl-leucine aldehyde