This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The S. pombe F-box protein Pof6 is essential for cell viability and contains an F-box in its amino-terminal portion, two Sec10 domains in the carboxy-terminal portion and a CAAX domain prior to the stop codon. Its closest homologue is Rcy1 from S. cerevisiae (24% identity), a non-essential F-box protein involved in protein recycling (which also contains a Sec10 domain and a CAAX domain). Pof6 has been shown to interact with Skp1 (the F-box-binding adaptor protein) consistently and, most of the residues important for the Skp1/F-box domain interface are conserved in the F-box of Pof6. Surprisingly, unlike other F-box proteins, Pof6 was found not to interact with the scaffold protein Cul1 raising the possibility that this F-box protein is part of a non-SCF complex. This hypothesis is supported by the fact that the residues important for the Cul1/F-box domain interface are not conserved in Pof6. To decipher the essential role for Pof6 in the cell, a TAP purification followed by MudPIT analysis was performed in collaboration with Dr. J. Thompson and Dr J. Yates, TSRI, San Diego. Among several new Pof6 interactors, a protein of high molecular weight, visible on purified silver-stained samples and well detected by mass spectrometry (139 unique peptides/792 total peptides) was identified. The gene encoding for this protein is essential in S. pombe and its interaction with Pof6 has been confirmed by coimmunoprecipitation. The function of this protein is now under investigation. The phenotypic characterization of the ts pof6 mutants, pof6-1166 and pof6-51 reveals similar defects for both strains: small sized cells with septation defects and an accumulation of cells containing 4C DNA content. These phenotypes suggest that DNA replication is not affected and that mutant cells are defective in cell growth rather than cell cycle progression. Altogether, the data collected so far lead us to the hypothesis that Pof6 function is related to the translation machinery as it seems to consistently interact with ribosome components, therefore its dysfunction would result in growth defects. As a result both pof6 ts strains appear to be sensitive to translation inhibitors, supporting the idea that Pof6 directly modulates the biogenesis or the activity of the ribosomes. Subsequently polysome profiling analysis (sucrose gradient centrifugation) showed that Pof6 co-sediments with the active polysomes, the 80S and 60S complexes. A portion of Skp1 co-migrates with Pof6, although most of the protein remains with smaller molecular weight complexes (i.e. SCF complexes). Inactivation of Pof6 modifies dramatically the polysome profile: the translating polysomes regressed and seem to accumulate into inactive mono-ribosomes, resulting in the increase of the 80S peak. This figure is characteristic of a diminution in translation activity. We are now planning to use the polysome profiling analysis to examine whether the Pof6 interactor co-sediments with Pof6 and Skp1 at the high molecular weight complexes, and investigate if Pof6 is involved in translation initiation and/or elongation.
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