Studies of the regulation of serine protease activity in the establishment of the dorsal-ventral axis of the Drosophila embryo
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Dorsal-ventral (DV) polarity in the Drosophila embryo is defined by spatially regulated activation of the transmembrane receptor Toll, which is uniformly distributed throughout the early embryo's plasma membrane. Ventral activation of Toll is accomplished through the local production of its activating ligand, a processed C-terminal fragment of the Spätzle protein, which is generated in the last step of a proteolytic cascade involving the sequentially-acting proteases Gastrulation Defective (GD), Snake and Easter. Pipe protein, a homologue of vertebrate glycosaminoglycan modifying enzymes, which is expressed during oogenesis in ventral follicle cells adjacent to the developing oocyte, is believed to control the ventrally restricted processing of Spätzle. pipe expression and the sulfation of its enzymatic target in the ventral follicle cells leads to the formation of a stable ventral cue, embedded in the eggshell. Recently the Pipe enzymatic target has been identified as several protein components of the vitelline membrane, the inner layer of the eggshell. Prior to this work, an important piece of information missing from our understanding of Drosophila DV patterning was the identity of the initial step in the protease cascade that requires Pipe activity. Here, I show that the processing of Snake is independent of Pipe activity, while the processing of Easter requires Pipe function, indicating that Easter processing by Snake is the key proteolytic step that is controlled by Pipe activity and presumably the first cleavage event that is spatially regulated. A second key gap in our understanding of Drosophila embryonic DV patterning concerned the role of GD in the protease cascade. While GD is the protease that cleaves and activates Snake, the existence of two distinct classes of complementing gd alleles has suggested that GD provides another, distinct function. Investigations described here indicate that the second function of GD is to promote the ability of activated Snake to process Easter, independent of its Snake-processing function. Finally, I provide evidence for the formation of protein complexes containing various components of the serine protease cascade, which suggest that conformational changes in the complexes, which act to promote productive interactions between the proteins, are an important aspect of their activation.