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A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands

2002; Springer Nature; Volume: 21; Issue: 16 Linguagem: Inglês

10.1093/emboj/cdf434

1460-2075

Siniša Urban,

Insect Resistance and Genetics

Article15 August 2002free access A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands Sinisa Urban Sinisa Urban MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Jeffrey R. Lee Jeffrey R. Lee MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Matthew Freeman Corresponding Author Matthew Freeman MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Sinisa Urban Sinisa Urban MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Jeffrey R. Lee Jeffrey R. Lee MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Matthew Freeman Corresponding Author Matthew Freeman MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Author Information Sinisa Urban1, Jeffrey R. Lee1 and Matthew Freeman 1 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK ‡S.Urban and J.R.Lee contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4277-4286https://doi.org/10.1093/emboj/cdf434 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Drosophila has three membrane-tethered epidermal growth factor (EGF)-like proteins: Spitz, Gurken and Keren. Spitz and Gurken have been genetically confirmed to activate the EGF receptor, but Keren is uncharacterized. Spitz is activated by regulated intracellular translocation and cleavage by the transmembrane proteins Star and the protease Rhomboid-1, respectively. Rhomboid-1 is a member of a family of seven similar proteins in Drosophila. We have analysed four of these: all are proteases that can cleave Spitz, Gurken and Keren, and all activate only EGF receptor signalling in vivo. Star acts as an endoplasmic reticulum (ER) export factor for all three. The importance of this translocation is highlighted by the fact that when Spitz is cleaved by Rhomboids in the ER it cannot be secreted. Keren activates the EGF receptor in vivo, providing strong evidence that it is a true ligand. Our data demonstrate that all membrane-tethered EGF ligands in Drosophila are activated by the same strategy of cleavage by Rhomboids, which are ancient and widespread intramembrane proteases. This is distinct from the metalloprotease-induced activation of mammalian EGF-like ligands. Introduction The epidermal growth factor (EGF) receptors are a family of receptor tyrosine kinases essential for the control of many cellular processes, including proliferation, survival and differentiation (Adamson, 1990; Schweitzer and Shilo, 1997; Domínguez et al., 1998; Sibilia et al., 1998). In Drosophila, EGF receptor signalling is used repeatedly and in many different contexts throughout growth and development, highlighting the need for stringent regulation of receptor activity (Schweitzer and Shilo, 1997; Wasserman and Freeman, 1997). Genetic and molecular studies have revealed that the production of an active EGF ligand by the signal-sending cell is a key regulatory step in receptor activation. In most contexts, the main activating ligand of the EGF receptor in Drosophila is Spitz, a membrane-tethered EGF ligand that resembles several of the mammalian EGF receptor ligands, including transforming growth factor-α (TGF-α) (Rutledge et al., 1992; Freeman, 1994; Tio et al., 1994; Schweitzer et al., 1995; Tio and Moses, 1997). Like its mammalian counterparts, Spitz is translated as a transmembrane molecule with an extracellular EGF domain. Full-length Spitz is unable to signal and must be processed into an active, soluble form. Rhomboid-1 and Star, first implicated by genetic analysis, are the primary positive regulators of Spitz activation in the signal-sending cell (Ruohola-Baker et al., 1993; Sturtevant et al., 1993; Freeman, 1994; Bang and Kintner, 2000; Wasserman et al., 2000; Lee et al., 2001; Urban et al., 2001; Klämbt, 2002; Tsruya et al., 2002). In the absence of Star, Spitz is confined to the endoplasmic reticulum (ER) whereas Rhomboid-1 is in the Golgi apparatus (Lee et al., 2001). Star relocalizes Spitz from the ER to the Golgi, where Spitz is cleaved within its transmembrane domain by Rhomboid-1, an intramembrane serine protease (Urban et al., 2001). Drosophila EGF receptor activation is thus controlled by the regulated intracellular trafficking and proteolytic activation of its ligand, Spitz (Lee et al., 2001; Urban et al., 2001; Tsruya et al., 2002). This mechanism defines a new pathway for growth factor release in which the cleavage occurs intracellularly and is a form of 'regulated intramembrane proteolysis' (RIP) (Brown et al., 2000; Huppert and Kopan, 2001). This is in contrast to all other known examples of growth factor activation, which use cell surface metalloproteases to release the active growth factor domains by a cleavage in the juxtamembrane region (Arribas et al., 1996; Black and White, 1998). Regulation of Drosophila EGF receptor ligand activation is made more complex by the fact that there are seven Rhomboids and three membrane-tethered EGF ligands (Guichard et al., 2000; Wasserman et al., 2000; Ghiglione et al., 2002). For example, despite its key role in the proteolytic cleavage of Spitz, there are several contexts where the Rhomboid-1 protease is not essential for EGF receptor activation. In the developing Drosophila eye, Spitz activation is required for the recruitment of all cell types, while Rhomboid-1 is completely dispensable in this process (Freeman et al., 1992). Instead, genetic analysis has shown that Rhomboid-3, a close homologue of Rhomboid-1, functions as an eye-specific Rhomboid to activate EGF receptor signalling (Wasserman et al., 2000). Recently, Rhomboid-2 (also called Brother of Rhomboid; Guichard et al., 2000) has been shown to trigger cleavage of Gurken in cell culture (Ghiglione et al., 2002). As well as Rhomboid-1-independent EGF receptor activation, there are several examples of Rhomboid-1-dependent EGF receptor signalling that do not depend on Spitz, most notably in the developing eye and wing (Simcox, 1997; Domínguez et al., 1998; Nagaraj et al., 1999). These imply the existence of a missing EGF receptor ligand, predicted to be a substrate for Rhomboid-1. There are two other membrane-tethered EGF receptor ligands in Drosophila: Gurken and Keren. Gurken function is restricted to oogenesis where it is required for polarizing the egg by signalling from the oocyte to the overlying EGF receptor-expressing follicle cells (Schüpbach, 1987; Neuman-Silberberg and Schüpbach, 1993; Gonzalez-Reyes et al., 1995). Recent evidence suggests that, like Spitz, Gurken requires post-translational activation to signal (Ghiglione et al., 2002). Consistent with a possible role in Gurken processing, Rhomboid-2 is expressed exclusively in the oocyte (Guichard et al., 2000). The third apparent membrane-tethered EGF receptor ligand, Keren (previously also called Spitz-2; Baonza et al., 2001) or Gritz (Flybase reference 0126856), is highly similar to Spitz and was identified originally by the Drosophila Genome Project. It is the best candidate for the Rhomboid-dependent ligand required for eye and wing development since it is the only other membrane-tethered EGF receptor ligand apparent in the Drosophila genome. However, there is no mutant allele to prove its biological function. Furthermore, although the missing ligand is predicted to be Rhomboid dependent (Wasserman et al., 2000), nothing is known about Keren processing. The multiplicity of Rhomboids and potential ligands in Drosophila implies that there is potential for complexity in EGF receptor signal processing. We have addressed four questions that arise from this possibility. First, do other Drosophila Rhomboids share Rhomboid-1's protease activity? Secondly, do all participate in EGF receptor signalling, or do they activate other pathways? Thirdly, do the different combinations of ligands, Rhomboids and Star produce different outcomes, indicating a potential for complex combinatorial control of EGF receptor signalling? Fourthly, is Keren a genuine ligand for the EGF receptor and thereby likely to be the missing ligand? To begin to answer these questions, we have analysed systematically the properties of all combinations of four Rhomboids and all three membrane-tethered EGF receptor ligands in the Drosophila EGF signal activation pathway. Our results show that all four Rhomboids have similar proteolytic activity and that all membrane-tethered ligands are substrates for the Rhomboid proteases. Although not required for ligand release from the cell in every Rhomboid–ligand combination, we found that Star acts as an ER export factor for all three ligands. Our data also suggest that at least these four Rhomboids are dedicated to regulation of EGF receptor activity in Drosophila and that Keren is indeed an effective EGF receptor ligand. These results indicate a common mechanism for activating all membrane-tethered EGF receptor ligands in Drosophila but demonstrate the potential for a complex system of regulation. Results Rhomboids 2, 3 and 4 are proteases that promote EGF receptor signalling Rhomboids 1–4 form a group of similar proteiins. Within the seven Rhomboid-like molecules in Drosophila, Rhomboids 1–4 are more closely related to each other than they are to any of the remaining three Rhomboids (Figure 1A). Rhomboids 6 and 7 are more divergent, although they both retain the residues required for the serine protease activity of Rhomboid-1 (Urban et al., 2001). Rhomboid-5 has similarities to the others but does not contain the catalytic residues. Because of their similarity to each other and as we have been unable to identify full-length cDNAs for Rhomboids 5, 6 or 7, we focused our analysis on Rhomboids 1–4. Figure 1.Drosophila Rhomboids 1–4 are proteases that can cleave Spitz. (A) A dendrogram illustrating the sequence relationship between the seven Drosophila Rhomboids. Rhomboids 1–4 are most similar to each other. (B) GFP-tagged Spitz was cleaved by Rhomboids 1–4 in a Star-dependent manner when transiently expressed in COS cells and analysed by western blotting; the cleaved N-terminus of Spitz accumulated in the medium in all four cases. Note that Rhomboids 2, 3 and 4 could cleave Spitz intracellularly in the absence of Star (small arrowhead, full-length Spitz indicated by large arrowhead: compare the relative levels of full-length and cleaved Spitz in the presence of each Rhomboid), but this cleaved product was not secreted. The white arrowhead shows the hyperglycosylated form of Spitz caused by Star expression in the absence of Rhomboid (Lee et al., 2001). (C) Rhomboid levels in the Spitz cleavage assay were reduced by decreasing the amount of transfected rhomboid DNA (shown in ng) (Urban et al., 2001). All four Rhomboids cleaved and secreted Spitz at equivalent levels, even when they became limiting. Download figure Download PowerPoint Rhomboids 1–4 catalyse Spitz proteolysis. We tested whether Rhomboids 1–4 all had proteolytic activity against Spitz, the known substrate for Rhomboid-1 (Lee et al., 2001; Urban et al., 2001) (Figure 1B). Spitz was cleaved efficiently by all Rhomboids tested, albeit with some significant differences. Our cell culture assay allowed us to distinguish Spitz cleaved in cell lysates from that which had been secreted into the medium. The amount of cleaved Spitz detected in cells varied with different Rhomboids; no or very little intracellular cleaved Spitz was detected in cells with Rhomboid-1, while cleaved intracellular Spitz was readily detected with Rhomboids 2–4. This is most apparent by comparing the relative levels of full-length and cleaved Spitz in cell lysates (Figure 1B; large and small arrowheads, respectively). In the presence of Star, the amount of secreted Spitz present in the medium was the same for all core Rhomboids, even when they were made limiting by reducing their levels of expression (Figure 1C), indicating that all four Rhomboids have similar levels of proteolytic activity against Spitz. Star is not essential for cleavage by Rhomboids 2, 3 and 4. Star regulates Spitz cleavage by Rhomboid-1 by transporting Spitz to the Golgi apparatus (Lee et al., 2001). Strikingly, although Star was essential for ligand secretion into the culture medium in each case, it did not affect the ability of Rhomboids 2, 3 and 4 to catalyse Spitz cleavage (Figure 1B). The extensive O-linked glycosylation that is diagnostic of transit through the Golgi apparatus (and which increases the apparent molecular weight of Spitz) (Lee et al., 2001) was not present in cell lysates [Figure 1B; compare the size of secreted green fluorescent protein (GFP)–Spitz in the media (∼55 kDa) with the size of the cleaved intracellular GFP–Spitz (∼43 kDa)]. Therefore, in contrast to Rhomboid-1, Rhomboids 2, 3 and 4 caused the accumulation of an intracellular cleaved Spitz that was not transported past the trans-Golgi network and thus not secreted. Rhomboids 2–4 promote EGF receptor signalling in Drosophila. The ability of Rhomboids 1–4 to catalyse Spitz cleavage in the tissue culture assay suggested that all may be involved in activating the EGF receptor in vivo. This has been clearly demonstrated for Rhomboid-1, was genetically determined in the case of Rhomboid-3, and was proposed for Rhomboid-2 (Ruohola-Baker et al., 1993; Sturtevant et al., 1993; Wasserman and Freeman, 1997; Guichard et al., 2000; Wasserman et al., 2000; Lee et al., 2001; Ghiglione et al., 2002). To investigate this further, we compared the potential activity of Rhomboids 2–4 in vivo by overexpressing them in developing Drosophila tissues. In all cases examined, Rhomboids 2–4 caused similar phenotypes to Rhomboid-1, consistent only with EGF receptor hyperactivation. When expressed in the developing wing, for example, all core Rhomboids produced ectopic and thickened vein phenotypes similar to those observed for Rhomboid-1 (Figure 2). This phenotype was modified predictably by mutations in other members of the EGF receptor pathway (data not shown). Furthermore, as in cell culture assays, all four Rhomboids were synergistic with the co-expression of Star (e.g. Rhomboid-4 shown in Figure 2). In all cases, UAS Rhomboids 1 and 3 produced consistently strong wing phenotypes, whereas Rhomboids 2 and 4 were weaker. Similar results were obtained in the eye, follicle cells of the ovary and the embryo (data not shown). Importantly, no other phenotypes were observed in eyes, wings or embryos expressing Rhomboids, suggesting that they do not affect any other pathways. If, for example, the previously uncharacterized Rhomboids 2 or 4 caused the activation of other signalling pathways, their ectopic expression would lead to additional phenotypes. These observations confirm that Rhomboids 2–4 contain the same proteolytic activity as Rhomboid-1; furthermore, the absence of phenotypes associated with other pathways strongly suggests that Rhomboids 1–4 are all dedicated to regulating EGF receptor signalling. Figure 2.Analysis of Rhomboid 1–4 activity in Drosophila. Rhomboids 1–4 were ectopically expressed in wings using the MS1096-Gal4 driver and caused EGF receptor hyperactivation phenotypes, including thick veins and blisters. Multiple transgenic lines were isolated in each case: Rhomboids 2 and 4 produced a spectrum of phenotypes (each indicated with three panels), which included phenotypes identical to Rhomboids 1 and 3, but their average phenotypes were significantly weaker. While the expression of Star and the weakest Rhomboid-4 transgene (which was X-linked and examined in females) yielded only subtle effects, if any, their co-expression resulted in strongly synergistic phenotypes (bottom row). Download figure Download PowerPoint These data demonstrate that Rhomboids 1–4 all share proteolytic activity against the ligand Spitz. The next question we addressed was whether the other Drosophila membrane-tethered ligands, Keren and Gurken, were also substrates for any of Rhomboids 1–4. Keren processing by Rhomboids and Star Keren is highly similar to Spitz. We identified a new Spitz-like gene as a cDNA submitted to GenBank by the Berkeley Drosophila cDNA sequencing project (DDBJ/EMBL/GenBank accession No. AA990660). With the subsequent completion of the Drosophila genome sequence, this gene has been annotated as Keren (Gadfly CG8056) and is the only previously unknown membrane-tethered EGF-like molecule identified by the Drosophila genome project. Keren has been referred to previously as Spitz-2 and Gritz. Like Spitz and Gurken, Keren has a single extracellular EGF repeat and a single transmembrane domain. The amino acid sequence of Keren is more closely related to Spitz than to Gurken (49% identity, 55% similarity to Spitz; 30% identity, 37% similarity to Gurken, Figure 3A). While all three ligands were predicted to have N- and O-linked glycosylation signals, Spitz contains a 10 residue insert in its N-terminus, which contains an additional O-linked glycosylation site. Consistent with this, Spitz is the only ligand to be hyperglycosylated in the presence of Star (compare Figure 1B, white arrow, with Figures 3B and 5A), although deletion of the insert does not fully abolish hyperglycosylation (data not shown). Figure 3.Keren processing by Star and Rhomboids 1–4. (A) A GCG pileup alignment of Keren, Gurken and Spitz. A dashed line indicates the locations of the EGF domains. Transmembrane domains predicted by TMHMM (Krogh et al., 2001) are indicated with a solid line. (B) Western blots of conditioned medium and lysates from COS cells transfected with Keren with or without Star and/or Rhomboids 1–4. GFP–Keren was expressed alone (−) or in the presence (+S) or absence (−S) of Star with each of the four Rhomboids (R1–R4). Keren was cleaved and secreted efficiently by all four Rhomboids in the presence of Star; it was also cleaved and secreted at a lower level by Rhomboids 3 and 4 in the absence of Star. An intracellular cleaved product (arrowhead) was produced by all four Rhomboids. (C) Processing of Keren by Star and Rhomboid-1 in S2 cells: compared with Spitz, there is a higher level of cleavage and secretion triggered by Star or Rhomboid-1 alone, but the presence of Rhomboid-1 and Star together enhanced processing. (D and E) Intracellular localization of Keren. Cells were transfected with GFP–Keren with or without Myc-Star. The localization of each protein was detected by immunofluorescence. (D) GFP–Keren co-localized with an antibody against an endogenous ER marker (αPDI). (E) Co-expression of Star with GFP–Keren caused Keren to be exported from the ER and accumulate in the Golgi apparatus and at the cell surface. Download figure Download PowerPoint Figure 4.Ectopic expression of Keren in wings and the ventral epidermis. (A) Wild-type wing. (B and C) Full-length UAS-Keren expressed in wings with the MS1096-Gal4 driver produced a range of EGF receptor hyperactivity phenotypes: thickened wing veins and extra wing vein material. (D–F) Expression of secreted Spitz in the embryo with the arm-Gal4 driver produced typical EGF receptor overactivation phenotypes in the ventral epidermis (Szüts et al., 1997; Payre et al., 1999). Wild-type denticle belts have a characteristic arrangement of six rows (D); overexpression of sSpi (E) or sKer (F) caused the formation of extra denticles. Download figure Download PowerPoint Rhomboids 1–4 cleave Keren. Keren's high similarity to Spitz suggested that it might also be a substrate for Rhomboids 1–4, and we have confirmed this: Rhomboids 1–4 all cleaved Keren in a mammalian tissue culture assay. There was, however, an interesting distinction between Keren and Spitz: Star was not essential for Keren secretion in every case (Figure 3B). A significant amount of Keren was secreted in the presence of Rhomboids 3 and 4 alone. Despite this, Star always enhanced the secretion of cleaved Keren, implying that it can interact with Keren. Another difference between Keren and Spitz was that cleaved but non-secreted Keren accumulated in cells in the presence of any of Rhomboids 1–4. Recall that this was the case for Spitz cleaved by Rhomboids 2–4, but not for Rhomboid-1. This discrepancy could be explained if Keren was a better substrate than Spitz for Rhomboids 1–4 or as a consequence of the higher expression levels of Keren. Keren processing by Rhomboid-1 in Drosophila S2 cells was similar but not identical to that observed in COS cells: in S2 cells, a small amount of Keren was secreted in the absence of additional Star and Rhomboid-1 (Figure 3C), but the amount increased in the presence of Star or Rhomboid-1 alone and, as in mammalian cells, was significantly enhanced in the presence of both Rhomboid-1 and Star (Figure 3C). Keren is localized to the ER. A key to understanding the regulation of Spitz activation by Rhomboid-1 and Star was the observation that the ligand was restricted to the ER in the absence of Star (Lee et al., 2001). We therefore examined the intracellular localization of Keren in COS cells. Like Spitz, Keren was only detectable in the ER; it exhibited characteristic perinuclear and reticular staining and co-localized with the ER marker protein disulfide isomerase (PDI) (Figure 3D). Star exports Keren out of the ER. Star's role in Spitz activation is to export Spitz from the ER to the Golgi apparatus where it encounters the proteolytic activity of Rhomboid-1. Star also promoted the release of Keren into the medium, suggesting its role was similar to that in Spitz processing. To test this directly, we co-expressed Star and Keren in COS cells. In the presence of Star, Keren was no longer detectable in the ER and was now entirely in the Golgi apparatus and plasma membrane (Figure 3E). This relocalization of Keren was very similar to the relocalization observed for Spitz, suggesting that Keren also needs to be relocalized to the Golgi apparatus for efficient processing and secretion. Keren can activate EGF receptor signalling in vivo To test the prediction that Keren is a genuine EGF receptor ligand, we misexpressed it in developing Drosophila tissues. By analogy to similar experiments with Spitz, we expressed either full-length, membrane-tethered Keren (mKeren), or a truncated form that corresponds to the extracellular, secreted form of Keren (sKeren). In most contexts, full-length Spitz is unable to signal when misexpressed because Star and Rhomboid-1 activity limit its activation, while a truncated form of Spitz, missing its transmembrane domain and C-terminus, signals in a Rhomboid-1- and Star-independent manner (Schweitzer et al., 1995). In contrast to Spitz, ectopic expression of mKeren activated the EGF receptor pathway in both eyes and wings. For example, when expressed in the developing wing, mKeren produced wing phenotypes similar to misexpression of other positively acting members of the EGF receptor pathway, ranging from thickened and ectopic wing veins to blistering (Figure 4B and C). In many cases, the activation was so strong that the entire wing was converted to vein-like material (Figure 4C). These results indicate that either Keren has membrane-tethered, juxtacrine activity or that it is processed and secreted, possibly by Rhomboids 3 or 4 (see Discussion), which would be consistent with the results obtained in the cell culture assay. Figure 5.Gurken processing by Star and Rhomboids 1–4. (A) Western blots of conditioned medium and lysates from COS cells transfected with Gurken with or without Star and/or Rhomboids 1–4. GFP–Gurken was expressed alone (−) or in the presence (+S) or absence (−S) of Star with each of the four Rhomboids (R1–R4). In COS cells, Gurken was cleaved and secreted by all four Rhomboids, independently of Star; all four also caused intracellular cleavage. (B) Processing of Gurken in S2 cells was similar to that in COS cells. (C and D) Intracellular localization of Gurken. Cells were transfected with GFP–Gurken and/or Myc-Star. The localization of each protein was detected by immunofluorescence. (C) GFP-tagged Gurken co-localized with an antibody against an endogenous ER marker (αPDI). (D) Co-expression of Star with GFP–Gurken caused Gurken to be exported from the ER and accumulate in the Golgi apparatus but, in contrast to Spitz and Keren, not at the cell surface. Download figure Download PowerPoint To test whether the activity of mKeren represented the full potential phenotype of ectopic Keren, or whether proteolytic activation had the potential to activate it further, we examined the effects of sKeren. This form was even more potent than mKeren, causing lethality even when driven by tissue-specific drivers. However, in the embryo, where mKeren had a weak effect, ubiquitous misexpression of sKeren caused lethality and resulted in significantly widened denticle belts and a reduction in naked cuticle in the ventral epidermis (Figure 4F), identical to that caused by the misexpression of sSpitz (Figure 4E) (O'Keefe et al., 1997; Szüts et al., 1997). The greater potency of sKeren therefore suggests that Keren is proteolytically activated in vivo. Together, these results indicate that Keren is a genuine ligand for the EGF receptor, being able to activate the receptor pathway in vivo. They also suggest that Keren is activated by Rhomboid proteases and Star, although it remains possible that the membrane-tethered form of the ligand has some juxtacrine activity. Gurken processing by Rhomboids and Star Rhomboids 1–4 cleave Gurken. The improved signalling ability of a secreted form of Gurken and the recent observation that Rhomboid-2 is required for Gurken activity in a wing misexpression assay (Guichard et al., 2000) raise the possibility that Gurken may also be processed by Rhomboid proteases. To investigate this further, we analysed Gurken processing by Rhomboids 1–4 in the mammalian cell culture assay. As with Spitz and Keren, we found that all core Rhomboids could indeed cleave Gurken, although the role of Star was more variable: in some experiments, Star appeared to have very little influence (Figure 5A and B), while in others it significantly enhanced secretion. In fact, Gurken cleavage appeared very efficient: unlike the other two ligands, only the cleaved form was seen in cell lysates. This parallels the complete cleavage of Gurken deduced in oocytes (Ghiglione et al., 2002) and suggests that Gurken may be the best substrate for Rhomboids 1–4. In COS cells, Gurken was secreted in response to each of Rhomboids 1–4 in the absence of Star. Although Star improved secretion of Gurken for Rhomboids 2 and 4, it had only a minor effect on Gurken secretion in the presence of Rhomboids 1 and 3 (Figure 5A). Gurken cleavage by Rhomboid-1 in S2 cells was similar to that observed in COS cells (Figure 5B). Gurken is localized to the ER. To determine if Gurken processing might also be regulated by its intracellular localization, we expressed GFP–Gurken in COS cells. Like Spitz and Keren, Gurken was confined to the ER and could not be detected in the Golgi apparatus (Figure 5C). Star exports Gurken out of the ER. Despite the more limited effect of Star on Gurken secretion, co-expression of Star caused the export of Gurken from the ER to the Golgi apparatus, implying that its trafficking can be regulated similarly to Spitz and Keren. Unlike for Spitz and Keren, however, in the absence of Rhomboids, Star did not cause full-length Gurken to relocate to the plasma membrane (Figure 5D). Instead, relocalized Gurken remained confined to the Golgi apparatus. This suggests that Gurken may lack signals required for Golgi to plasma membrane transport, or may contain signals for Golgi retention. In either case, it suggests that post-Golgi secretion of Gurken may be a regulated process. Subcellular localization of Rhomboids 1–4 Rhomboid-1 is a Golgi-localized protein and its proteolytic activity for Spitz is confined to this compartment; it does not cleave Spitz to any significant degree in the ER. To determine whether the amount of Spitz cleavage in the absence of Star correlated with the subcellular localization of Rhomboids 1–4 in COS cells, we examined their intracellular localization by immunofluorescence (Figure 6). Rhomboids 2 and 3 were indistinguishable from Rhomboid-1: they were observed solely in the Golgi apparatus. In contrast, Rhomboid-4 was present at high levels at the plasma membrane, as well as in the Golgi apparatus. None of Rhomboids 1–4 were detectable in the ER. Figure 6.Intracellular localization of HA-tagged Rhomboids 1–4 in COS cells. Rhomboids 2 and 3 could only be detected in the Golgi apparatus like Rhomboid-1, and co-localized with the known Golgi marker p115. Rhomboid-4 displayed a pronounced cell surface distribution, although it was also detected in the Golgi apparatus, but not in the ER. Download figure Download PowerPoint ER-cleaved Spitz cannot be secreted Unlike Spitz cleavage by Rhomboid-1, all other Rhomboids led to substantial intracellular accumulation of cleaved Spitz and concomitant reduction of the full-length form. The distinct glycosylation signature of Spitz allows its location to be inferred (Lee et al., 2001): the cleaved ligand appeared to be in a pre-trans-Golgi compartment. This intracellular cleavage suggests that there might be a low but functional amount of Rhomboid in the E