Identification of a conserved F-box protein 6 interactor essential for endocytosis and cytokinesis in fission yeast.
The F-box domain is a degenerated motif consisting of approximately 40 amino acid residues that specifically bind Skp1, a core component of the SCF (Skp1-Cdc53/Cullin 1-F-box protein) ubiquitin ligase. Recent work, mainly performed in budding yeast, indicates that certain F-box proteins form non-SCF complexes together with Skp1 in the absence of cullins and play various roles in cell cycle and signalling pathways. However, it is not established whether these non-SCF complexes are unique to budding yeast or common in other eukaryotes. In the present paper, using TAP (tandem affinity purification) coupled to MudPIT (Multidimensional Protein Identification Technology) analysis, we have identified a novel conserved protein, Sip1, in fission yeast, as an interacting partner of an essential F-box protein Pof6. Sip1 is a large HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor)-repeats containing protein (217 kDa) and forms a complex with Pof6 and Skp1. This complex does not contain cullins, indicating that it is a novel non-SCF complex. Like Pof6 and Skp1, Sip1 is essential for cell viability and temperature-sensitive sip1 mutants display cell division arrest as binucleate cells with septa. Sip1 localizes to the nucleus and dynamic cytoplasmic dots, which are shown in the present study to be endocytic vesicles. Consistent with this, sip1 mutants are defective in endocytosis. Furthermore, towards the end of cytokinesis, constriction of the actomyosin ring and dissociation of type II myosin and septum materials are substantially delayed in the absence of functional Sip1. These results indicate that the conserved Sip1 protein comprises a novel non-SCF F-box complex that plays an essential role in endocytosis, cytokinesis and cell division.
Jourdain
Isabelle
I
Cancer Research UK, London Research Institute, UK.
Spielewoy
Nathalie
N
Thompson
James
J
Dhut
Susheela
S
Yates
John R
JR
Toda
Takashi
T
D000483:Alleles; D002467:Cell Nucleus / Q000378:metabolism; D048749:Cytokinesis / Q000235:genetics / Q000502:physiology*; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D020043:Genes, Essential; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D044783:F-Box Proteins; D005656:Fungal Proteins; C473775:Pof6 protein, S pombe; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D049452:Green Fluorescent Proteins