A39
Functional analysis of an essential F-box protein in fission yeast
Nathalie Spielewoy1, James Thompson2, John R. Yates III2, Takashi Toda1
1Cancer Research UK, London, UK, 2The Scripps Research Institute, La Jolla, CA, USA
The F-box proteins have been well described as substrate-binding components of the SCF complexes (Skp1/Cullin/F-box). In this context, they play the crucial role of substrate recognition in a specific and timely manner targeting them to degradation via the proteasome.
My work focuses on identifying the essential role of Pof6, an F-box protein proposed to form a non-SCF complex (Hermand D. et al., 2003). By TAP purification coupled to MuPIT analysis, I confirmed the interaction between Pof6 and Skp1 and identified a novel Pof6’s interactor named Sip1 for Pof Six Interacting Protein 1. A Sip1-TAP purification and a set of coimmunoprecipitations confirmed the interactions between the three proteins. Like Pof6 and Skp1, the uncharacterised protein Sip1 is essential in fission yeast and conserved among the eukaryotes.
The characterisation of a sip1 thermosensitive allele, sip1-62 ts, reveals defects in cytokinesis; G2 synchronised sip1-62 ts cells die at the onset of telophase with two nuclei properly separated by an abnormally thick septa correctly formed and positioned. In parallel, an essential component of the actomyosin ring, myosin II, appears delocalised.
Such data are consistent with the cellular localisation of GFP-Sip1 to the nuclei and trafficking cytoplasmic vesicles that can gather around the contractile ring. By performing time-lapse studies of GFP-Pof6, we also identified a specific and transient accumulation of the protein at the medial zone just before septum appearance.
Altogether the data suggest that Sip1 and Pof6 play an essential role at the equatorial zone during cytokinesis in S. pombe.
