Proteolysis in Caenorhabditis elegans sex determination: cleavage of TRA-2A by TRA-3

Abstract

The Caenorhabditis elegans tra-3 gene promotes female development in XX hermaphrodites and encodes an atypical calpain regulatory protease lacking calcium-binding EF hands. We report that despite the absence of EF hands, TRA-3 has calcium-dependent proteolytic activity and its proteolytic domain is essential for in vivo function. We show that the membrane protein TRA-2A, which promotes XX female development by repressing the masculinizing protein FEM-3, is a TRA-3 substrate. Cleavage of TRA-2A by TRA-3 generates a peptide predicted to have feminizing activity. These results indicate that proteolysis regulated by calcium may control some aspects of sexual cell fate in C. elegans.

Figure 1

Regulation of sexual fate in C. elegans focusing on the role of tra-3. (A) Simplified genetic pathway of somatic sex determination (Meyer 1997; Kuwabara 1999; and references therein). Wild-type XX diploids are hermaphrodites, which are self-fertile females that produce first sperm, then oocytes, and XO diploids are males. The pathway consists of a cascade of negative regulatory interactions, whereby an upstream gene inhibits the activity of its downstream neighbor. The state of each gene (high/low) during XX or XO development is indicated. The decision to adopt a male or female somatic fate is determined by tra-1. The pathway has been modified to indicate that tra-2 is epistatic to tra-3 (this work). (B) Molecular model of the signal transduction pathway underlying the genetic pathway of somatic sex-determination. In XX hermaphrodites, TRA-2A is depicted as a multipass membrane protein that binds and represses FEM-3 through its carboxyl terminus, possibly by sequestration. We propose that TRA-3 is a protease that cleaves and potentiates the activity of TRA-2A (this work); however, the fate of the carboxy-terminal TRA-2A peptide is unknown. TRA-3 is present in both sexes (S. Sokol and P. Kuwabara, unpubl.).

Figure 2

The C. elegans sex-determining protein TRA-3 catalyzes calcium-dependent autolysis in vitro. (A) Schematic representation of amino-terminally myc-tagged wild-type TRA-3 and catalytically inactive TRA-3(C83S) expressed in vitro. The catalytic triad of Cys, His, and Asn is marked in white. The four domains of TRA-3 are labeled I–III, and T. (B) Wild-type TRA-3 incubated in the absence (−) or presence (+) of 5 mm calcium (lanes 1,2); catalytically inactive TRA-3(C83S) incubated in the absence (−) or presence (+) of 5 mm calcium (lanes 3,4). Full-length TRA-3 (∼76 kD); (arrow) TRA-3 cleavage product (∼50 kD). (C) Predicted site of TRA-3 autolysis based on sizes of wild-type and truncated TRA-3 cleavage products (S. Sokol and P. Kuwabara, unpubl.).

Figure 3

Expression and immunological detection of myc-tagged TRA-3 and TRA-2A in Sf9 cells. (A) Schematic representation of wild-type TRA-3 and catalytically inactive TRA-3(C83S) proteins expressed in Sf9 cells (see legend to Fig. 2A). (B) Immunoblot of Sf9 extracts expressing TRA-3 or TRA-3(C83S) probed with anti-myc (lanes 1,2). (C) Immunoblot of membrane-enriched Sf9 extracts expressing myc-tagged TRA-2A probed with anti-TRA-2. (D) Localization of TRA-2A to the plasma membrane of Sf9 cells using anti-TRA-2. Note that cell at right expresses TRA-2A more strongly than cell at left because of variability in infection efficiency.

Figure 4

Proteolytic cleavage of TRA-2A by TRA-3. (A) Immunoblot probed with anti-TRA-2 of Sf9 cell extracts expressing TRA-2A alone (lane 1), coexpressing TRA-2A and wild-type TRA-3 (lane 2), or coexpressing TRA-2A and catalytically inactive TRA-3(C83S) (lane 3). (Open arrowhead) Full-length TRA-2A (∼170 kD); (solid arrowhead) carboxy-terminal TRA-2A proteolytic peptide (∼55 kD). (B) Immunoblot from A stained with Ponceau S to show that wild-type and inactive TRA-3 are comparably expressed. (C) Proposed relationship between TRA-2A/B and TRA-2A cleavage peptide. Predicted site of TRA-2A cleavage by TRA-3 is indicated by arrowhead.

Figure 5

Detection of somatic feminization in XX and XO animals using a vit-2::gfp reporter. (A) Nomarski DIC photomicrograph of an adult XX him-8; crEx65 hermaphrodite. (B) The same animal in A examined by epifluorescence to reveal intestinal GFP expression. (C) Nomarski DIC photomicrograph of an adult XO him-8; crEx65 male following heat shock. (D) The same animal in C examined by epifluorescence to reveal ectopic intestinal GFP fluorescence, which is indicative of somatic feminization. In all panels: anterior, left; ventral, down. Scale bar, 10 μm.