Components of an SCF ubiquitin ligase localize to the centrosome and regulate the centrosome duplication cycle.
Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle.
Freed
E
E
Departments of Pathology and Microbiology and Immunology, Stanford University School of Medicine, Palo Alto, California 94305 USA.
Lacey
K R
KR
Huie
P
P
Lyapina
S A
SA
Deshaies
R J
RJ
Stearns
T
T
Jackson
P K
PK
D016475:3T3 Cells; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002502:Centrioles / Q000502:physiology / Q000648:ultrastructure; D018385:Centrosome / Q000201:enzymology* / Q000648:ultrastructure; D006224:Cricetinae; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; D005260:Female; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D000075687:NEDD8 Protein; D010063:Ovum; D010453:Peptide Synthases / Q000378:metabolism*; D016196:S Phase; D044786:S-Phase Kinase-Associated Proteins; D044843:SKP Cullin F-Box Protein Ligases; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000502:physiology / Q000648:ultrastructure; D014020:Tissue Extracts / Q000502:physiology; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism; D014982:Xenopus laevis
C080443:CDC4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; C513273:FBXW7 protein, human; C513272:Fbxw7 protein, mouse; D000075687:NEDD8 Protein; C513172:Nedd8 protein, mouse; D044786:S-Phase Kinase-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D014020:Tissue Extracts; D014452:Ubiquitins; C577449:CDC34 protein, S cerevisiae; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D010453:Peptide Synthases