Cell Cycle–Regulated Attachment of the Ubiquitin-Related Protein Sumo to the Yeast Septins

Abstract

SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins. SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis. We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis. Septins are components of a belt of 10-nm filaments encircling the yeast bud neck. Intriguingly, only septins on the mother cell side of the bud neck are sumoylated. We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED). Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G2/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle. This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites. Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle.

Some assembly required: yeast septins provide the instruction manual

“…After completion of cytokinesis and separation of mother and daughter cells, the septin rings disassemble for a short period in G1. Whether disassembly involves the degradation of septin subunits or whether septins are recycled has not been determined, but a role for a specific post-translational modification (SUMOylation) by small ubiquitin-like modifier (SUMO) has been proposed [62]. In fact, septins are, by far, the most abundant substrates for SUMOylation in budding yeast.…”

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

“…Protein precipitates were then resuspended in 2X SDS-loading buffer, heated for 5 min and subjected to SDS-PAGE. To purified His6-tagged proteins, extracts were prepared in guanidine as described [13]. Cleared extracts were made 10 mM in imidazole and continuously applied to a 1 ml Ni His-Trap column (GE Healthcare) at room temperature overnight.…”

“…These proteins share a RINGfinger related sequence motif, the SP-RING domain, with the PIAS1 (Protein Inhibitor of Activated Signal transducer and activator of transcription) protein of human cells [11,12]. The Siz1 and Siz2 ligases are active on septins and may have partially overlapping specificities [10,13,14]. Together, Siz1 and Siz2 account for 90% of the total sumoylation in yeast, and cells lacking both SIZ1 and SIZ2 are viable but slow growing [9,10].…”

In vivo modeling of polysumoylation uncovers targeting of Topoisomerase II to the nucleolus via optimal level of SUMO modification

“…First, for the absolute majority of proteins, only a tiny fraction of molecules is modified (Johnson 2004), so that purification of the modified protein and a biochemical assessment of its function is extremely difficult. For the same reason, direct physical mapping of sumoylated sites and therefore their mutagenesis are rarely attempted (Johnson and Blobel 1999).…”