Multiple domains in Siz SUMO ligases contribute to substrate selectivity

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Saccharomyces cerevisiae possesses Siz1, Siz2/Nfi1, and Mms21 as known SUMO E3 ligases.
• The substrate specificity of SUMO ligases is not fully understood, unlike the high specificity of ubiquitin ligases.

Purpose of the Study:

• To investigate the substrate specificity of Siz1 and Siz2 SUMO E3 ligases in Saccharomyces cerevisiae.
• To determine the factors influencing substrate selectivity among Siz proteins.

Main Methods:

• In vivo sumoylation assays were performed.
• Analysis of Siz1 domains involved in substrate targeting and localization.
• Investigated sumoylation of specific proteins including PCNA, Prp45, and septins (Cdc3, Cdc11, Shs1/Sep7, Cdc10, Cdc12).

Main Results:

• Siz1 and Siz2 exhibit unique substrates, yet both can stimulate sumoylation of many common substrates.
• Residual sumoylation occurs in the absence of Siz1 and Siz2, with some dependence on Mms21.
• Siz1 utilizes distinct domains (N-terminal PINIT, C-terminal) for targeting different substrates like PCNA, Prp45, and septins.
• Septins lacking native SUMO sites can be sumoylated by Siz1 when fused to a SUMOylation motif.

Conclusions:

• Substrate selectivity by Siz proteins is mainly determined by their local concentration and cellular localization.
• Distinct domains within Siz1 mediate targeting to specific substrates and localization.
• The findings provide insights into the regulatory mechanisms of SUMOylation in yeast.