Cullin neddylation and substrate-adaptors counteract SCF inhibition by the CAND1-like protein Lag2 in Saccharomyces cerevisiae.
Cullin-based E3 ubiquitin ligases are activated through covalent modification of the cullin subunit by the ubiquitin-like protein Nedd8. Cullin neddylation dissociates the ligase assembly inhibitor Cand1, and promotes E2 recruitment and ubiquitin transfer by inducing a conformational change. Here, we have identified and characterized Lag2 as a likely Saccharomyces cerevisiae orthologue of mammalian Cand1. Similar to Cand1, Lag2 directly interacts with non-neddylated yeast cullin Cdc53 and prevents its neddylation in vivo and in vitro. Binding occurs through a conserved C-terminal beta-hairpin structure that inserts into the Skp1-binding pocket on the cullin, and an N-terminal motif that covers the neddylation lysine. Interestingly, Lag2 is itself neddylated in vivo on a lysine adjacent to this N-terminal-binding site. Overexpression of Lag2 inhibits Cdc53 activity in strains defective for Skp1 or neddylation functions, implying that these activities are important to counteract Lag2 in vivo. Our results favour a model in which binding of substrate-specific adaptors triggers release of Cand1/Lag2, whereas subsequent neddylation of the cullin facilitates the removal and prevents re-association of Lag2/Cand1.
Siergiejuk
Edyta
E
ETH Zürich, Institute of Biochemistry, Schafmattstrasse 18, Zürich, Switzerland.
Scott
Daniel C
DC
Schulman
Brenda A
BA
Hofmann
Kay
K
Kurz
Thimo
T
Peter
Matthias
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D044842:Cullin Proteins / Q000737:chemistry* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry
C100988:Cdc53 protein, S cerevisiae; D044842:Cullin Proteins; C101054:LAG2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases