In the C. elegans hermaphrodite, first sperm then oocytes are produced within the same somatic gonad. Based on genetic and molecular analysis, the genes
gld-1 and
fog-2 are required to promote male sex determination in the hermaphrodite germ line by the down regulation of
tra-2 , thus allowing for a transient period of spermatogenesis (Schedl and Kimble, 1988; Francis et al. , 1995; Jan et al. , 1999; Clifford et al. , 2000). GLD-1 directly binds the
tra-2 3’UTR causing its translational repression. The FOG-2 protein binds GLD-1 and is associated with the
tra-2 3’UTR in a ternary complex, but unlike GLD-1, FOG-2 does not make direct contact with the
tra-2 mRNA. The FOG-2 protein contains two domains; an N-terminal F-box motif and a novel C-terminal 200 amino acid putative protein-protein interaction domain. Traditionally, F-box motif containing proteins have been associated with the ubiquitin mediated degradation pathway. Canonical F-box proteins interact with Skp1, a core component of SCF ( s
kp1/ c
dc53/ F -box, E3 ubiquitin ligase) complex, via their N-terminal F-box and recruit specific substrates for ubiquitin modification via their C-terminal protein-protein interaction domains. The functional definition of an F-box requires that it contain the minimal sequence elements required to interact with the SCF core component Skp1. Therefore, we are conducting a directed screen of all Skp1 homologs in the C. elegans genome using FOG-2 as bait. Interestingly, FOG-2 is able to bind F46A9.5 and F46A9.4, which share the highest overall identity to the defining member yeast Skp1 (44% and 39% respectively). This suggests that the F-box motif in FOG-2 is functional and therefore could play a role in the ubiquitin mediated degradation of specific substrates involving the SCF and 26S proteasome. However, RNAi phenotypes of the aforementioned Skp1 homologs were embryonic lethal so that FOG-2 related functions in sex determination remain unclear. To define the broad scope of FOG-2 interactions, a C. elegans two-hybrid cDNA library (R. Barstead) was screened. The C. elegans Mcb1 homolog was identified as a FOG-2 interacting protein. RNAi analysis of C. elegans Mcb1 resulted in feminization of the hermaphrodite germ line and partial suppression of the
fem-3(
q20gf) phenotype, which is analogous to what is observed for
gld-1 and
fog-2 lf mutations. This provides in vivo support for an interaction between C. elegans Mcb1 and FOG-2. In S. cerevisiae, Mcb1 is a nonessential protein that co-purifies with the 19S regulatory cap of the 26S proteasome, binds multiubiquitin chains in vitro, and plays an ancillary role in substrate-specific protein turnover (van Nocker et al. , 1996). While the yeast Mcb1 protein has been implicated in protein turnover, the C. elegans Mcb1 homolog is unlikely to be involved in GLD-1 degradation as, similar to FOG-2, the Mcb1 homolog appears to promote hermaphrodite spermatogenesis. When RNAi of the Mcb1 homolog is performed in a strain carrying GLD-1::GFP the feminizing phenotype is recapitulated but changes the GLD-1 accumulation or degradation pattern are not observed. Thus, the C. elegans Mcb1 homolog promotes hermaphrodite spermatogenesis and may represent another component of the FOG-2/GLD-1/
tra-2 3'UTR translational control complex. We propose two working models for FOG-2 as a scaffold in recruiting proteins such as Mcb1 into the framework of GLD-1 binding the
tra-2 3'UTR and mediating its translational regulation: 1) FOG-2, perhaps in concert with Mcb1, degrades a positive regulator of
tra-2 translation. 2) FOG-2 and/or Mcb1 are involved in the ubiquitin modification, but not degradation, of another component(s) of the
tra-2 3'UTR ternary complex. A role for F-box proteins in the direct translational regulation of a specific mRNA represents a novel function for this family of proteins.