Sub-cellular localization of a potential tumor suppressor, the F-Box protein FBXO47

Abstract

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The cellular level of many oncoproteins and tumor suppressors is controlled by the ubiquitin system. In eukaryotes, the SCF ubiquitin-ligase complex performs much of the targeted proteins ubiquitylation. F-box proteins are critical components of the SCF and are involved in substrate recognition and recruitment for ubiquitination and consequent degradation by the proteasome. We have previously identified a novel member of F-Box proteins FBXO47 (GeneBank#AY700575). The gene is located at 17q12 band, region deleted in various cancer such as breast, gastric, prostate, ovarian, and kidney cancer. Human FBXO47 is composed of 11 exons and code for a 453 amino acids protein. This gene is preferentially expressed in normal tissue as opposed to corresponding tumor tissue. Although these results suggest a potential tumor suppressor role for FBXO47, it function are not know. To elucidate the role of FBXO47, we first determined its sub-cellular localization. A GFP plasmid was construct using cDNA FBXO47 synthesized from testis total RNA. RT-PCR product was purified, and inserted to pEGFP-N3 plasmid. The construction was then transfected in COS-7 cells. Mitochondria were stained with Mitotracker Red, and nuclei were labeled with TOPRO-3. Localization of the FBXO47 protein in COS-7 cells was observed using a Leica TSC-SP1 microscope . The confocal data indicated that FBXO47-GFP fusion protein is located in mitochondria, and the Metamorph program confirmed the co-localization of fluorescent labels. This result suggests a role for FBXO47 in the regulation of mitochondrial protein. To better understand the relations between mitochondria and this putative tumor suppressor gene, identification of FBXO47 protein partners is necessary. For this purpose, we are at present using the BacterioMatch two-hybrid vector using different variants of FBXO47 as baits, and a commercially available kidney cDNA library. This should give us important information for the functional understanding of this large family of F-Box proteins.