bride of sevenless

Promoter Structure

bride of sevenless contains a TATA-box deficient (TATA-less) promoter. Such promoters have a conserved sequence motif (A/GGA/TCGTG) termed the downstream promoter element (DPE), located about 30 nucleotides downstream of the RNA start site of many TATA-less promoters, including bride of seveless. DNase I footprinting of the binding of epitope-tagged TFIID to TATA-less promoters reveals that the factor protects a region that extends from the initiation site sequence (about +1) to about 35 nucleotides downstream of the RNA start site. There is no such downstream DNase I protection induced by TFIID in promoters with TATA motifs. This suggests that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters (Burke, 1996).

Protein Interactions

A direct interaction between BOSS and Sevenless has been demonstrated by the heterotypic aggregation of cell lines expressing both proteins. In the developing eye the Sevenless-dependent internalization of BOSS by the R7 precursor cell provides evidence for a direct interaction between these two proteins in vivo (Kramer, 1991).

The seven pass transmembrane domain of BOSS is necessary for its function; a soluble form of BOSS, consisting of the extracellular domain, acts as an antagonist of the SEV receptor both in vivo and in vitro (Hart, 1993a).

The entire Boss protein, from its extreme N-terminus to its extreme C-terminus, is internalized by sev-expressing tissue culture cells and by the R7 precursor cell in the developing eye imaginal disc. The receptor-mediated transfer of a transmembrane ligand represents a novel mechanism for protein transfer between developing cells (Cagan, 1992).

The Boss protein from D. virilis (Bossvir) retains strong amino acid identity with Boss from D. melanogaster (Bossmel): 73% identity in the N-terminal extracellular domain and 91% identity in the seven-transmembrane domain, including the cytoplasmic tail. The Bossvir genes are able to rescue the DM boss1 mutation, and the expression of Bossvir protein in DM is indistinguishable from that of Bossmel protein. The predicted SEV protein from DV (Sevvir) is 63% identical to SEV from DM (Sevmel). A chimeric gene, (sevvir/mel), encoding the extracellular domain of Sevvir and the cytoplasmic domain of Sevmel rescues the DM sevd2 mutation through interaction with either Bossvir or Bossmel (Hart, 1993b).

Mutations in the hook gene inhibit endocytosis of the BOSS transmembrane ligand into multivesicular bodies. Hook is a novel cytoplasmic protein that has the capacity dimerize mediated by a coiled-coil domain. A connection between the function of Hook and the actin cytoskeleton is provided by the visible bristle phenotype of hook mutants. Mutations in the forked, chickadee and singed genes cause malformations of bristles similar to hook mutations. Chickadee and Singed are fly homologs of the actin-binding proteins profilin and fascin, required for the formation of actin bundles in the early stages of extension of the bristle shaft (Krämer, 1996).

Eukaryotic cells carefully regulate trafficking of internalized proteins. In most cells, endocytic vesicles deliver internalized cargo to early endosomes from which many proteins are shuttled back to the plasma membrane through a recycling compartment. Other proteins accumulate in vacuolar subcompartments of early endosomes before their transformation into mature multivesicular bodies (MVBs) named for their characteristic morphology. From MVBs, endocytic cargo is delivered to morphologically distinct prelysosomal structures, the multilammelar late endosomes. The biochemical mechanisms that regulate these trafficking events late in the endocytic pathway are not well understood. Mutations in Drosophila constitute a resource for the genetic dissection of endocytic trafficking in multicellular organisms. Notably, the discovery that the shibire gene encodes the Drosophila homolog of Dynamin is important for revealing Dynamin function. Early work on the shibire gene suggested its role in pinching endocytic vesicles from the plasma membrane, a hypothesis recently confirmed by detailed biochemical analysis. A direct effect of the shibire mutation on receptor-mediated endocytosis was first demonstrated for the Bride of Sevenless (Boss) ligand. A search for additional mutations that altered internalization of Boss has revealed a role for the Drosophila hook gene in endocytic trafficking. At the light microscopy level, the effects of hook and shibire mutations appeared similar: the amount of detectable Boss protein in R7 cells is reduced, when compared with wild type. However, an analysis of a viable hook null allele reveals considerable differences in the functional consequences of hook and shibire mutations. For example, the shibire mutation caused paralysis and defects in cell-cell communication during development, processes unaffected by a complete loss of hook function. The hook gene encodes a cytoplasmic dimeric protein of 679 amino acids with an extended central coiled coil domain, which is conserved in two human homologs. Immunohistochemical studies reveal that Hook localizes to endocytic vesicles and large vacuoles that are distinct from lysosomes. This localization indicates that Hook may function late in endocytic trafficking. To better understand the role of Hook, the specific step in endocytosis altered by the hook mutation has been sought (Sunio, 1999 and references).

To dissect the intracellular trafficking of internalized ligands in Drosophila a new approach has been developed to visualize the pathway taken by the Bride of Sevenless (Boss) ligand after its internalization into R7 cells. A chimeric protein consisting of horseradish peroxidase (HRP) fused to Boss (HRP-Boss) was expressed in R8 cells. This chimera is fully functional: it rescues the boss mutant phenotype, and its trafficking is indistinguishable from that of the wild-type Boss protein. The HRP activity of the chimera was used to follow HRP-Boss trafficking on the ultrastructural level through early and late endosomes in R7 cells. In both wild-type and hook mutant eye disks, HRP-Boss is internalized into R7 cells. In wild-type tissue, Boss accumulates in mature multivesicular bodies within R7 cells; such accumulation is not observed in hook eye disks, however. Quantitative electron microscopy reveals a loss of mature MVBs in hook mutant tissue compared with wild type, whereas more than twice as many multilammelar late endosomes are detected. The genetic analysis indicates that Hook is required late in endocytic trafficking to negatively regulate delivery from mature MVBs to multilammelar late endosomes and lysosomes (Sunio, 1999).

Multiple mechanisms could explain the effect of hook mutations on mature MVBs and late endosomes. A model in which the wild-type Hook protein is directly required for the stability of mature MVBs by inhibiting their fusion to multilammelar late endosomes or lysosomes is favored. It is proposed that hook mutations result in an increased rate of transport of internalized ligands to multilammelar late endosomes causing premature degradation of ligands. This model is consistent with the following four observations. (1) Hook is detected on endocytic vesicles and vacuoles but not lysosomes by indirect immunofluorescence. (2) Internalized Boss is not detected in hook mutant R7 cells, although no change is detected in the internalization of HRP-Boss as determined by its HRP activity. (3) hook mutations alter endocytic trafficking for all internalized ligands tested. (4) The increase of multilammelar late endosomes or lysosomes in the absence of mature MVBs in hook mutant tissue points to increased lysosomal transport. The hook mutant phenotype is reminiscent of the cellular defects in cells derived from mucolipidosis type IV patients that exhibit a dramatic increase in multilammelar endocytic structures and an increased transport rate of internalized lipids to lysosomes (Sunio, 1999 and references).

Although the HRP-Boss transgene is a useful tool to study late stages in Boss endocytosis, it also provides information about the uptake of the Boss transmembrane ligand across cell boundaries. The observations argue that Boss trans-endocytosis occurs through the uptake of small patches of R8 membrane into R7 cells, rather than a phagocytic process. Internalization of Boss is preceded by its clustering on the R8 cell membrane, visualized by patches of membrane stained with HRP activity. Oligomerization of Boss-Sevenless complexes into higher-order complexes has previously been evoked to explain the lack of Sevenless activation by monomeric and dimeric Boss ligands. Such clustering might effectively exclude other transmembrane proteins from the small membrane patches that are being pulled into the R7 cell by receptor-mediated endocytosis. The results with Boss are consistent with the observations of trans-endocytosis of other transmembrane proteins. The Delta, Serrate, and Lag-2 transmembrane ligands are trans-endocytosed after binding to receptors of the Notch class. For Delta trans-endocytosis, phagocytosis could be ruled out, because independent cell surface markers are not cointernalized. A similar mechanism of trans-endocytosis might also explain the internalization of the homotypic cell adhesion protein apCam (Sunio, 1999 and references).

By screening for Drosophila mutants exhibiting aberrant Bride of sevenless (Boss) staining patterns on eye imaginal disc epithelia, a point mutation was recovered in Hsc70-4, the closest homolog to bovine clathrin uncoating ATPase. Hsc70 is a constitutively expressed member of the Hsp70 protein family that has been implicated in many processes including folding of newly synthesized polypeptides, translocation of proteins across the endoplasmic reticulum, stabilizing proteins under stress conditions, and antigen presentation. In vitro, Hsc70 promotes the release of clathrin triskelions and other coat proteins from CCVs by binding to clathrin and thus disrupting the clathrin cage concomitant with ATP hydrolysis. Like other members of the Hsp70 protein family, Hsc70 has a low intrinsic ATPase activity, which can be stimulated by cofactors. The relevant cofactor in the uncoating reaction is thought to be auxilin, a member of the DnaJ protein family characterized by a conserved J-domain motif. In addition, auxilin contains a clathrin binding domain, suggesting that auxilin first binds to CCVs, and then recruits ATP-bound Hsc70 proteins via its J-domain. The J-domain interaction stimulates Hsc70 ATPase activity, thereby stabilizing the binding of Hsc70 to clathrin, and driving triskelion dissociation. After uncoating, Hsc70 remains associated with the soluble pool of clathrin (Chang, 2002 and references therein).

Although the mutant Hsc70-4 allele is lethal, analysis of mutant clones generated by FLP/FRT recombination demonstrates that the Sevenless-mediated internalization of Boss is blocked in mutant Hsc70-4 eye disc epithelial cells. Endocytosis of other probes is also greatly inhibited in larval Garland cells. Immunostaining and EM analysis of the mutant cells revealed disruptions in the organization of endosomal/lysosomal compartments, including a substantial reduction in the number of clathrin-coated structures in Garland cells. The Hsc70-4 mutation also interactes genetically with a dominant-negative mutant of dynamin, a gene required for the budding of clathrin-coated vesicles (CCVs). Consistent with these phenotypes, recombinant mutant Hsc70 proteins exhibit diminished clathrin uncoating activity in vitro. Together, these data provide genetic support for the long-suspected role of Hsc70 in clathrin-mediated endocytosis, at least in part by inhibiting the uncoating of CCVs (Chang, 2002).

Although these experiments provide genetic proof of a role for Hsc70 in endocytosis, some important questions still remain. The link between uncoating as measured in vitro with the block in endocytosis observed in vivo must remain somewhat correlative because it is not possible to directly measure clathrin uncoating activity per se in intact cells. It is highly likely, however, that the impaired uncoating activity of Hsc70^R447H in vitro reflects a defect in the enzyme's role in regulating clathrin function within the cell. This conclusion is strongly supported by the observed genetic interaction between Hsc70-4 and dynamin and the profound effects of Hsc70-4^R447H on clathrin distribution in cells. Hsc70 is known to bind to the soluble pool of clathrin, and Hsc70-clathrin complexes are defective in self-assembly. Thus, in addition to its role in clathrin disassembly, Hsc70 may function as a chaperone to stabilize and/or maintain the function of the soluble pool of clathrin. Indeed, the nearly complete loss of identifiable CCVs and/or clathrin-coated pits in Garland cells expressing the mutant Hsc70-4 allele is also consistent with a severe disruption in the regulation of clathrin assembly/disassembly in these mutant cells. Regardless, these observations provide strong evidence for a physiological role for Hsc70 in clathrin-mediated endocytosis (Chang, 2002).

Recent work has suggested that Hsc70 may function not just at the internalization step, but at multiple steps of the endocytic pathway, including receptor recycling. Conceivably, this situation reflects a role for clathrin in recycling from endosomes, a possibility consistent with persistent observations of clathrin-coated buds apparently emanating from endosomal tubules. In addition, in polarized epithelial cells, the AP-1B clathrin adaptor complex plays a role in ensuring basolateral recycling during endocytosis. However, it is possible that Hsc70 has other functions in addition to clathrin uncoating. Staining of Hook, a cytosolic protein associated with early endosomes, is disrupted in Hsc70 mutant cells, suggesting that Hsc70 may have a role in properly organizing endosomal compartments, although it is not yet clear how the Hk protein itself is associated with membranes. Furthermore, a novel J-domain protein, Rme8, has recently been shown to participate in endocytosis in C. elegans. Although there is no evidence documenting direct interaction between Rme8 and Hsc70, the identification of another J-protein in the endocytic pathway certainly raises the possibility that the mechanism of Hsc70 function in endocytosis might be more complicated than previously envisioned. With the continued development of stage-specific assays for individual events during endocytic and biosynthetic membrane traffic in Drosophila cells, the availability of an Hsc70-4 mutant with endocytic phenotypes will prove useful in elucidating the nature of the steps under its direct or indirect control (Chang, 2002).

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