Worm Breeder's Gazette 12(3): 30 (June 15, 1992)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

tra-3 is a Pain

Tom Barnes

Department of Molecular Biology, MGH, Boston, MA 02114

In a previous Gazette (12#1, p.35), I reported the molecular characterization of the sex determination gene tra-3 .Genetic analysis suggests that tra-3 acts as a positive regulator of tra-2 .The tra-2 -encodedproduct ( Tra-2 p)resembles an integral membrane protein (Kuwabara and Kimble, 1989 Meeting), and is the putative receptor for Her-1 p(Hunter and Wood, Nature 1992). Tra-3 presembles calpain, a calcium-regulated cytosolic protease that is found in vertebrates and invertebrates. Many roles have been suggested for calpains in vertebrates, but most involve processing of substrates (either activation or inactivation) near membranes and in response to signal transduction events that elevate calcium levels. Processing of Tra-2 pwould fit neatly with the two-domain model of Tra-2 pstructure (Kuwabara et al. 1991 Meeting); the activation of Tra-2 pby cleavage between the domains might follow simply from the increased diffusion of the cytosolic domain, improving its putative sequestration functions of the putative Fem-123 pcomplex. This model is consistent with the available genetic data, but other models are possible.

Calpains purify as heterodimers, which associate non-covalently through related calcium-binding regions. The intact heterodimer is inactive, and becomes active on calcium addition resulting in subunit dissociation. The large subunit is catalytic, while the small is regulatory ( Tra-3 presembles catalytic subunits). Both subunits appear to be highly modular. The large subunit has four 'domains': an ~70 amino-acid, charged N-terminal domain that is largely cleaved off upon protease activation; a 250-residue protease domain, which is reasonably similar to the papain/actinidin/cathepsin class of cysteine proteases; a 200-residue domain of as yet no demonstrated function (but possibly involved with membrane/ phospholipid association); and finally, the C-terminal 180-residue calcium-binding domain containing four putative 'EF hand' motifs. The small subunit has an extremely similar 180 residue domain at its C-terminus, with the intron positions in these domains in the two genes being totally preserved. The positions of the C-termini in all calpains are strongly conserved. The N-terminus of the small subunit extends for some 50 amino acids, and contains a poly(Gly) stretch before the EF hand domain. Calpains are evolutionarily ancient, being found in Schistosomes, Nematodes and Vertebrates.

At the last report (WBG 12#1, p.35), the 3' end of tra-3 had not been sequenced, as it lay outside the rescuing region. I have now sequenced an extra 1.2 kb downstream of the rescuing region, and report that there is no domain IV (EF hands) in Tra-3 p;instead there is a slightly shorter sequence. Interestingly, however, the C-terminal 3 amino acids correspond to a calpain terminus. It thus seems that tra-3 may have been derived from a canonical calpain, with the EF hands having diverged rapidly. Tra-3 pis probably therefore constitutive, which again is consistent with genetic data, and also probably exists as a monomer. (The possibility of calcium regulation of tra-3 activity always posed somewhat of a logistical problem.)

In certain male/female strains, tra-3 function can be made dispensable. The possibility thus arises that tra-3 function may be evolutionarily recent, and may not be shared by male/ female species. In contrast to this notion, however, is the fact that probing C. briggsae (male/ hermaphrodite), C. remnanei (male/female) and C. vulgarersis (male/female) genomic DNAs with tra-3 cDNA in a low stringency Southern revealed discrete cross-hybridizing bands in each; bands from C. elegans DNA were fully accounted for by tra-3 .Since this probing did not detect the other C. elegans calpain ( cm21 d11 ;LG I right subcluster; cDNA sequencing project), it suggests that the bands in the other species are more similar to tra-3 than tra-3 is to the other C. elegans calpain locus. The sequence available for cm21 d11 shows that in a region particularly divergent in calpains, cm21 d11 is much more closely related to vertebrate calpains than is tra-3 ,suggesting that cm21 d11 may be the (a?) real homologue of vertebrate calpains.

Literature Cited:

Kuwabara and Kimble, 1989 Meeting

Hunter and Wood, Nature 1992