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Studies on substrate recognition by the budding yeast separase.

Matt Sullivan1, Nadine C D Hornig, Thomas Porstmann

  • 1Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.

The Journal of Biological Chemistry
|October 31, 2003
PubMed
Summary

This study explores how budding yeast separase recognizes its substrates during mitosis. Separase cleaves cohesin proteins to separate sister chromatids. Researchers used mutagenesis to identify a cleavage site motif in Scc1. They found this motif in over 1,000 yeast proteins but could not confirm new substrates. Using a peptide inhibitor, they showed separase interacts with Scc1 even when the cleavage site is blocked. This suggests separase uses both the cleavage site and additional features for recognition. The findings highlight the complexity of separase-substrate interactions during anaphase.

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Area of Science:

  • Cell cycle regulation in eukaryotic biology
  • Protease substrate specificity in molecular genetics
  • Mitotic progression mechanisms in yeast systems

Background:

The mechanism of sister chromatid separation during anaphase relies on separase cleaving cohesin subunits. Prior research has shown that separase cleaves Scc1 in budding yeast. However, the exact sequence requirements for cleavage remain unclear. No prior work had resolved how separase distinguishes between substrates. This gap motivated a detailed analysis of cleavage site motifs. Researchers already know that separase interacts with multiple proteins during mitosis. Yet, the role of motifs beyond cleavage sites in substrate recognition is uncertain. This uncertainty drove the current investigation into motif specificity. The study aims to clarify whether cleavage site motifs alone are sufficient for separase recognition.

Purpose Of The Study:

This study aimed to determine the sequence requirements for separase cleavage of Scc1. The researchers focused on one of the two cleavage motifs in Scc1. They used systematic mutagenesis to identify critical residues. The goal was to derive a cleavage site consensus motif. The study also sought to test whether this motif is present in other yeast proteins. Researchers wanted to assess if proteins with the motif are separase substrates. The investigation examined if additional features beyond the motif influence recognition. The purpose was to clarify how separase interacts with its substrates.

Keywords:
separase cleavage siteScc1 motif analysisyeast mitotic regulationprotease substrate specificity

Frequently Asked Questions

Budding yeast separase recognizes the motif (not(FKRWY))(ACFHILMPVWY)(DE)X(AGSV)R/X, derived from systematic mutagenesis of Scc1.

The motif is found in 1,139 of 5,889 predicted yeast proteins, according to the study's analysis.

The inhibitor was used to block the cleavage site and test if separase still interacts with Scc1, revealing recognition beyond the motif.

The researchers could not confirm new separase substrates but observed other mitotic regulation in some proteins.

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Main Methods:

The researchers performed systematic mutagenesis on a cleavage site motif in Scc1. They analyzed the sequence requirements at this motif to identify conserved residues. Based on mutagenesis data, they derived a cleavage site consensus motif. The motif was used to screen predicted yeast proteins for potential substrates. They identified 28 candidate proteins containing the motif and 35 with a core (DE)XXR motif. Researchers tested these proteins for separase cleavage activity. They also examined if separase interacts with Scc1 when the cleavage site is blocked. A peptide inhibitor was used to assess interactions outside the cleavage site.

Main Results:

The cleavage site consensus motif was derived as (not(FKRWY))(ACFHILMPVWY)(DE)X(AGSV)R/X. This motif was found in 1,139 of 5,889 predicted yeast proteins. The researchers tested 28 candidate proteins with the motif and 35 with a core (DE)XXR motif. They could not confirm new separase substrates in these proteins. However, they observed other forms of mitotic regulation in some proteins. When a peptide inhibitor occupied the cleavage site, separase still interacted with Scc1. This suggests that separase recognizes features beyond the cleavage site motif. The results indicate that both the motif and additional determinants mediate substrate recognition.

Conclusions:

The authors propose that separase recognizes both a cleavage site consensus motif and additional features. They suggest that the motif alone is not sufficient for substrate recognition. The study shows that separase can interact with Scc1 even when the cleavage site is blocked. This implies that separase uses multiple determinants for substrate interaction. The findings indicate that motif presence does not guarantee separase cleavage. The researchers observed mitotic regulation in some candidate proteins. However, no new substrates were confirmed in this study. The results highlight the complexity of separase-substrate recognition mechanisms.

The study suggests separase recognizes both the cleavage site motif and features outside the motif for substrate interaction.

The authors propose that separase uses multiple determinants for substrate recognition, not just the cleavage site motif.