|Ph.D Student||Raveh-Barak Hadas|
|Subject||Structure-Function Analysis of the Cell-Fusion Protein EFF-1|
|Department||Department of Biology||Supervisor||Professor Benjamin Podbilewicz|
|Full Thesis text|
Cell-cell fusion is an essential process for many organisms. In C. elegans nematode the embryonic development depends on cell-fusion and mostly mediated by the membrane-protein EFF-1. The crystal structure of EFF-1’s extracellular-region was solved and shows a remarkable homology to the fold of class II viral membrane-fusion proteins. EFF-1, as the viral proteins, forms trimers, but it lacks a hydrophobic fusion-loop which is typical to its viral counterparts, and instead it has a more hydrophilic ‘cd-loop’. The viral fusion-proteins act in a unidirectional way from the viral membrane, but EFF-1 is required in both fusing membranes. Thus, while their structure is conserved, they mediate membrane-fusion using different mechanisms.
In order to examine EFF-1-mediated fusion mechanism and the significance of specific residues and regions for the protein function, we have compared the ability of wild-type-EFF-1 and different mutants to fuse cells. We have transfected mammalian cells, which normally do not fuse, with eff-1 wild-type or mutants, and evaluated their fusion rate.
One group of mutations was originally identified in worms and we tested it in cell-culture. All the tested EFF-1 mutations reduced EFF-1-mediated fusion in cells, and three out of four resulted in similar degrees of fusion-inhibition as in worms. These results strengthen the reliability of the heterologous cell system. One of these mutations, C135Y(hy32), eliminates the formation of a disulfide-bond which is conserved in all structurally-solved class II fusion-proteins and involves a cysteine located at the cd-loop. The membrane-expression of this mutant was aberrant and it was not functional. Thus, cysteine-135 and the cd-loop region are essential for EFF-1 function; maybe they are required for the correct folding and location, or maybe directly affect EFF-1's function.
A second group of mutations was planned together with our collaborators to interfere with the stability of EFF-1’s trimer. These mutations involve four residues which are either located at the core of the trimer or lead to an additional glycosylation that interferes with oligomerization. According to their results these mutations did impair the trimerization of EFF-1. We have constructed five combinations of these missense mutations; when expressed on the plasma-membrane of mammalian cells, these mutations reduced the level of EFF-1-mediated fusion. The effect of triple and quadruple combinations of mutations was more significant, both on trimerization and on fusion, compared to the single mutations. Thus, it is reasonable to conclude that EFF-1 trimerization is an essential step for EFF-1 function.