Studies on substrate recognition by the budding yeast separase.
Sister chromatid cohesion is resolved at anaphase onset when separase, a site-specific protease, cleaves the Scc1 subunit of the chromosomal cohesin complex that is responsible for holding sister chromatids together. This mechanism to initiate anaphase is conserved in eukaryotes from budding yeast to man. Budding yeast separase recognizes and cleaves two conserved peptide motifs within Scc1. In addition, separase cleaves a similar motif in the kinetochore and spindle protein Slk19. Separase may cleave further substrate proteins to orchestrate multiple cellular events that take place during anaphase. To investigate substrate recognition by budding yeast separase we analyzed the sequence requirements at one of the Scc1 cleavage site motifs by systematic mutagenesis. We derived a cleavage site consensus motif (not(FKRWY))(ACFHILMPVWY)(DE)X(AGSV)R/X. This motif is found in 1,139 of 5,889 predicted yeast proteins. We analyzed 28 candidate proteins containing this motif as well as 35 proteins that contain a core (DE)XXR motif. We could so far not confirm new separase substrates, but we have uncovered other forms of mitotic regulation of some of the proteins. We studied whether determinants other than the cleavage site motif mediate separase-substrate interaction. When the separase active site was occupied with a peptide inhibitor covering the cleavage site motif, separase still efficiently interacted with its substrate Scc1. This suggests that separase recognizes both a cleavage site consensus sequence as well as features outside the cleavage site.
Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Matt
M
Sullivan
Nadine C D
NC
Hornig
Thomas
T
Porstmann
Frank
F
Uhlmann
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000705:Anaphase; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry*; D002868:Chromosomal Proteins, Non-Histone; D010450:Endopeptidases / Q000737:chemistry*; D018386:Kinetochores / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D008938:Mitosis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D004718:Saccharomycetales / Q000201:enzymology*; D064247:Separase; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C490529:MCD1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; C437104:Slk19 protein, S cerevisiae; D010450:Endopeptidases; C577986:ESP1 protein, S cerevisiae; D064247:Separase