The human proteins FEZ1 (fasciculation and elongation protein zeta 1) and FEZ2 are orthologs of the protein UNC-76 from C. elegans, involved in the growth and fasciculation of the worms axon. Pull down assays showed that the protein FEZ1 interacts with other proteins (e.g., the protein SCOCO, short coiled-coil protein), mitochondria, and vesicles. These components may therefore represent cargoes to be transported along the microtubule, and the transport may be mediated through FEZ1 reported binding to kinesins (KIF3A). We previously showed that FEZ1 dimerizes in its N-terminal region and interacts with other proteins, including the candidate cargoe proteins, through its C-terminus. Here, we studied the fragment FEZ1(92-194) as well as full-length 6xHis-FEZ1 (1-392) in vitro and endogenous FEZ1 isolated from HEK 293 cells and were able to demonstrate the formation of an intermolecular disulfide bond through FEZ1 Cys-133, which appears to be essential for dimerization. This disulfide bond may be important for the FEZ1 role as a dimeric and bivalent transport adaptor molecule, since it establishes a strong link between the monomers, which could be a prerequisite for the simultaneous binding of two cargoes.
BackgroundThe FEZ (fasciculation and elongation protein zeta) family designation was purposed by Bloom and Horvitz by genetic analysis of C. elegans unc-76. Similar human sequences were identified in the expressed sequence tag database as FEZ1 and FEZ2. The unc-76 function is necessary for normal axon fasciculation and is required for axon-axon interactions. Indeed, the loss of UNC-76 function results in defects in axonal transport. The human FEZ1 protein has been shown to rescue defects caused by unc-76 mutations in nematodes, indicating that both UNC-76 and FEZ1 are evolutionarily conserved in their function. Until today, little is known about FEZ2 protein function.Methodology/Principal FindingsUsing the yeast two-hybrid system we demonstrate here conserved evolutionary features among orthologs and non-conserved features between paralogs of the FEZ family of proteins, by comparing the interactome profiles of the C-terminals of human FEZ1, FEZ2 and UNC-76 from C. elegans. Furthermore, we correlate our data with an analysis of the molecular evolution of the FEZ protein family in the animal kingdom.Conclusions/SignificanceWe found that FEZ2 interacted with 59 proteins and that of these only 40 interacted with FEZ1. Of the 40 FEZ1 interacting proteins, 36 (90%), also interacted with UNC-76 and none of the 19 FEZ2 specific proteins interacted with FEZ1 or UNC-76. This together with the duplication of unc-76 gene in the ancestral line of chordates suggests that FEZ2 is in the process of acquiring new additional functions. The results provide also an explanation for the dramatic difference between C. elegans and D. melanogaster unc-76 mutants on one hand, which cause serious defects in the nervous system, and the mouse FEZ1 -/- knockout mice on the other, which show no morphological and no strong behavioural phenotype. Likely, the ubiquitously expressed FEZ2 can completely compensate the lack of neuronal FEZ1, since it can interact with all FEZ1 interacting proteins and additional 19 proteins.
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