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Multivalent Interactions between a Ubiquitin Ligase and its Substrates Mediate their Recruitment to Liquid Membrane‐less Organelles
Author(s) -
Mittag Tanja,
Bouchard Jill,
Martin Erik,
Otero Joel,
Marada Suresh,
Ogden Stacey
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.916.3
Subject(s) - ubiquitin ligase , organelle , ubiquitin , chemistry , biophysics , microbiology and biotechnology , dna ligase , dna , biology , biochemistry , gene
Membrane‐less organelles are specialized cellular compartments that concentrate a specific set of components within them, although they are not enclosed by membranes. Recently, it was recognized that they behave like liquid droplets, and the model put forward that they form through the process of liquid‐liquid phase separation (LLPS). LLPS mediates infinitely cooperative transitions and could therefore encode functional switches in biology. However, examples, in which enzymes undergo LLPS to turn activity on or off in a switch‐like fashion, are currently lacking. Here, we will address the question why the Speckle‐type POZ protein (SPOP), a substrate adaptor of a Cullin3‐RING ubiquitin ligase, is localized in a variety of different liquid membrane‐less organelles in the nucleus, including nuclear speckles, PML bodies and DNA damage loci. SPOP is, however, never found diffuse in the nucleus, raising the question which molecular mechanism leads to its localization. SPOP has two dimerization domains, which facilitate its self‐association into linear, higher‐order oligomers. The MATH domain, a substrate‐binding domain, interacts with linear motifs in substrates, so‐called “SPOP binding motifs” (SB motifs) and facilitates their recruitment to the ubiquitin ligase. Interestingly, many substrates contain several SB motifs, rendering substrate and ligase multivalent for each other. We demonstrate that SPOP and substrate undergo LLPS in a multivalency‐dependent fashion, and form liquid droplets in which both proteins are highly concentrated. Higher‐order SPOP oligomers promote substrate ubiquitination more strongly than self‐association deficient SPOP mutants. Our results lead to a model, in which SPOP and substrate localize to liquid organelles due to their ability to undergo LLPS together, generating hot spots of SPOP‐mediated ubiquitination. A change in substrate concentration should disassemble these ubiquitination factories cooperatively. SPOP mutations associated with a variety of cancers interfere with LLPS, suggesting a new molecular mechanism for loss of function mutations of tumor suppressors. SPOP may have evolved to specifically target substrates in membrane‐less organelles. Support or Funding Information This work was funded by a V Foundation Scholar Grant (T.M.), R01GM112846 (T.M.), the National Cancer Institute Cancer Center Support Grant P30CA21765 (at St. Jude Children's Research Hospital), and the American Lebanese Syrian Associated Charities (ALSAC).