Components Of Ubiquitin Ligase Complexes Research Articles

Cloning and Characterization of a Novel Oocyte-Specific Gene Encoding an F-Box Protein in Rainbow Trout (Oncorhynchus mykiss).

Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified a novel transcript which is represented by multiple ESTs derived only from the oocyte cDNA library. Analysis of tissue distribution of the novel transcript by reverse transcription polymerase chain reaction revealed that the novel gene is only expressed in unfertilized oocytes but not in 10 somatic tissues examined. Northern blot analysis showed the transcript size is approximately 2 kb. The full-length cDNA sequence for the novel gene was obtained by assembly of sequences from the EST clones and a 5'end sequence from a 5'RACE product. The complete cDNA is 1966 bp in length containing an open reading frame which encodes a predicted protein of 514 amino acids. The amino acid sequence does not share homology with any known proteins in the NCBI databases. However, a search of the Pfam protein database revealed that the protein contains an F-box motif at the N-terminus, indicating that this novel oocyte-specific protein is a member of the rapidly expanding family of F-box proteins. F-box proteins are components of ubiquitin-ligase complexes, in which they bind substrates for uiquitin-mediated proteolysis. F-box proteins have also been associated with cellular functions such as signal transduction and regulation of the cell cycle. Real time PCR analysis of the mRNA expression of the novel gene during vitellogenesis and early embryogenesis demonstrated that the expression of the novel gene is extremely high during early previtellogensis and decreases dramatically after fertilization. This result is confirmed by in situ hybridization which showed that the novel transcript is highly abundant in early previtellogenic oocytes. Western blot analysis using antibodies generated against a synthetic peptide of the novel protein detected a protein band of ~51 kDa in a protein sample prepared from oocytes. The specific expression of this gene in oocyte and its developmental pattern of expression suggest a role for this novel gene in the regulation of early oocyte.and follicle development. (poster)

The role of proteolysis in R gene mediated defence in plants

SUMMARY Within the last 10 years, numerous R genes have been cloned from natural genetic variation in model as well as crop plants, and these have been classified according to their motifs. Some of the downstream signalling components have also been identified by artificial mutagenesis. Recently, cloning of three of these signalling genes (COI1, RAR1 and SGT1b) from Arabidopsis, barley and tobacco have helped uncover the physiological link between defence signalling and ubiquitin-mediated protein degradation. The physical association of COI1 and SGT1b with the components of ubiquitin-ligase complexes has been shown. In addition, post-transcriptional silencing of some of the subunits of the ubiquitin-ligase complex has led to a loss of resistance, indicating that protein degradation may also act as a regulatory mechanism in plant defence. Over the next few years, we should expect to see more examples of the interplay between the defence response and protein degradation in plants.

Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein.

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins-ySKP1, CDC53 (Cullin), and the F-box protein CDC4-that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, we isolated hSKP1 in a two-hybrid screen with hCUL1, the closest human homologue of CDC53. Here, we show that hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins.