Intrinsically disordered substrates dictate SPOP subnuclear localization and ubiquitination activity

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Speckle-type POZ protein (SPOP) acts as a ubiquitin ligase adaptor, targeting substrates for proteasomal degradation.
• Most SPOP substrates feature multiple SPOP-binding (SB) motifs, enabling liquid-liquid phase separation (LLPS) with SPOP.
• Pancreatic and duodenal homeobox 1 (Pdx1), an insulin transcription factor, is degraded by SPOP, but only one SB motif was previously identified.

Purpose of the Study:

• To investigate the interaction mode between Pdx1 and SPOP.
• To determine if Pdx1 undergoes LLPS with SPOP, unlike other known substrates.
• To elucidate how Pdx1's interaction with SPOP influences SPOP's function and localization.

Main Methods:

• Identification and characterization of SPOP-binding motifs in Pdx1.
• Cell-based assays to assess liquid-liquid phase separation between Pdx1 and SPOP.
• Microscopy techniques to observe the subnuclear relocalization of SPOP upon interaction with Pdx1.
• Analysis of SPOP-mediated ubiquitination of Pdx1.

Main Results:

• A second SB motif was identified in the Pdx1 C-terminal region (Pdx1-C).
• Despite two SB motifs, Pdx1 did not undergo LLPS with SPOP in cells.
• Pdx1 interaction induced SPOP relocalization from nuclear speckles to the nucleoplasm.
• Efficient Pdx1 ubiquitination and turnover required both identified SB motifs.

Conclusions:

• Pdx1's interaction with SPOP does not lead to phase separation, diverging from typical SPOP substrates.
• Substrate-specific properties can direct the subnuclear localization of SPOP-substrate interactions and ubiquitination.
• The findings offer insights into the regulation of SPOP-mediated protein degradation based on substrate characteristics.