A Conserved Di-Basic Motif of Drosophila Crumbs Contributes to Efficient ER Export.

The Drosophila type I transmembrane protein Crumbs is an apical determinant required for the maintenance of apico-basal epithelial cell polarity. The level of Crumbs at the plasma membrane is crucial, but how it is regulated is poorly understood. In a genetic screen for regulators of Crumbs protein trafficking we identified Sar1, the core component of the coat protein complex II transport vesicles. sar1 mutant embryos show a reduced plasma membrane localization of Crumbs, a defect similar to that observed in haunted and ghost mutant embryos, which lack Sec23 and Sec24CD, respectively. By pulse-chase assays in Drosophila Schneider cells and analysis of protein transport kinetics based on Endoglycosidase H resistance we identified an RNKR motif in Crumbs, which contributes to efficient ER export. The motif identified fits the highly conserved di-basic RxKR motif and mediates interaction with Sar1. The RNKR motif is also required for plasma membrane delivery of transgene-encoded Crumbs in epithelial cells of Drosophila embryos. Our data are the first to show that a di-basic motif acts as a signal for ER exit of a type I plasma membrane protein in a metazoan organism.

Lipids trigger a conformational switch that regulates signal recognition particle (SRP)-mediated protein targeting.

Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.

Dominant renin gene mutations associated with early-onset hyperuricemia, anemia, and chronic kidney failure.

Through linkage analysis and candidate gene sequencing, we identified three unrelated families with the autosomal-dominant inheritance of early onset anemia, hypouricosuric hyperuricemia, progressive kidney failure, and mutations resulting either in the deletion (p.Leu16del) or the amino acid exchange (p.Leu16Arg) of a single leucine residue in the signal sequence of renin. Both mutations decrease signal sequence hydrophobicity and are predicted by bioinformatic analyses to damage targeting and cotranslational translocation of preprorenin into the endoplasmic reticulum (ER). Transfection and in vitro studies confirmed that both mutations affect ER translocation and processing of nascent preprorenin, resulting either in reduced (p.Leu16del) or abolished (p.Leu16Arg) prorenin and renin biosynthesis and secretion. Expression of renin and other components of the renin-angiotensin system was decreased accordingly in kidney biopsy specimens from affected individuals. Cells stably expressing the p.Leu16del protein showed activated ER stress, unfolded protein response, and reduced growth rate. It is likely that expression of the mutant proteins has a dominant toxic effect gradually reducing the viability of renin-expressing cells. This alters the intrarenal renin-angiotensin system and the juxtaglomerular apparatus functionality and leads to nephron dropout and progressive kidney failure. Our findings provide insight into the functionality of renin-angiotensin system and stress the importance of renin analysis in families and individuals with early onset hyperuricemia, anemia, and progressive kidney failure.

Signal peptide peptidase (SPP) assembles with substrates and misfolded membrane proteins into distinct oligomeric complexes.

SPP (signal peptide peptidase) is an aspartyl intramembrane cleaving protease, which processes a subset of signal peptides, and is linked to the quality control of ER (endoplasmic reticulum) membrane proteins. We analysed SPP interactions with signal peptides and other membrane proteins by co-immunoprecipitation assays. We found that SPP interacts specifically and tightly with a large range of newly synthesized membrane proteins, including signal peptides, preproteins and misfolded membrane proteins, but not with all co-expressed type II membrane proteins. Signal peptides are trapped by the catalytically inactive SPP mutant SPPD/A. Preproteins and misfolded membrane proteins interact with both SPP and the SPPD/A mutant, and are not substrates for SPP-mediated intramembrane proteolysis. Proteins interacting with SPP are found in distinct complexes of different sizes. A signal peptide is mainly trapped in a 200 kDa SPP complex, whereas a preprotein is predominantly found in a 600 kDa SPP complex. A misfolded membrane protein is detected in 200, 400 and 600 kDa SPP complexes. We conclude that SPP not only processes signal peptides, but also collects preproteins and misfolded membrane proteins that are destined for disposal.

Requirement of the Dynein-Adaptor Spindly for Mitotic and Post-Mitotic Functions in Drosophila.

Spindly was originally identified as a specific regulator of Dynein activity at the kinetochore. In early prometaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. Here, we present loss- and gain-of-function studies of Spindly using transgenic RNAi in Drosophila. Knock-down of Spindly in the female germ line results in mitotic arrest during embryonic cleavage divisions. We investigated the requirements of Spindly protein domains for its localisation and function, and found that the carboxy-terminal region controls Spindly localisation in a cell-type specific manner. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells, we altered Spindly protein levels in migrating cells and found that ovarian border cell migration is sensitive to the levels of Spindly protein. Our study uncovers novel functions of Spindly and a differential, functional requirement for its carboxy-terminal region in Drosophila.

Membrane topology of the Drosophila OR83b odorant receptor.

By analogy to mammals, odorant receptors (ORs) in insects, such as Drosophila melanogaster, have long been thought to belong to the G-protein coupled receptor (GPCR) superfamily. However, recent work has cast doubt on this assumption and has tentatively suggested an inverted topology compared to the canonical N(out) - C(in) 7 transmembrane (TM) GPCR topology, at least for some Drosophila ORs. Here, we report a detailed topology mapping of the Drosophila OR83b receptor using engineered glycosylation sites as topology markers. Our results are inconsistent with a classical GPCR topology and show that OR83b has an intracellular N-terminus, an extracellular C-terminus, and 7TM helices.

Leptin down-regulates insulin action through phosphorylation of serine-318 in insulin receptor substrate 1.

Insulin resistance in skeletal muscle is found in obesity and type 2 diabetes. A mechanism for impaired insulin signaling in peripheral tissues is the inhibition of insulin action through serine phosphorylation of insulin receptor substrate (Irs) proteins that abolish the coupling of Irs proteins to the activated insulin receptor. Recently, we described serine-318 as a protein kinase C (PKC)-dependent phosphorylation site in Irs1 (Ser-318) activated by hyperinsulinemia. Here we show in various cell models that the adipose hormone leptin, a putative mediator in obesity-related insulin resistance, promotes phosphorylation of Ser-318 in Irs1 by a janus kinase 2, Irs2, and PKC-dependent pathway. Mutation of Ser-318 to alanine abrogates the inhibitory effect of leptin on insulin-induced Irs1 tyrosine phosphorylation and glucose uptake in L6 myoblasts. In C57Bl/6 mice, Ser-318 phosphorylation levels in muscle tissue were enhanced by leptin and insulin administration in lean animals while in diet-induced obesity Ser-318 phosphorylation levels were already up-regulated in the basal state, and further stimulation was diminished. In analogy, in lymphocytes of obese hyperleptinemic human subjects basal Ser-318 phosphorylation levels were increased compared to lean individuals. During a hyperinsulinemic euglycemic clamp, the increment in Ser-318 phosphorylation observed in lean individuals was absent in obese. In summary, these data suggest that phosphorylation of Ser-318 in Irs1 mediates the inhibitory signal of leptin on the insulin-signaling cascade in obese subjects.

The Crumbs_C isoform of Drosophila shows tissue- and stage-specific expression and prevents light-dependent retinal degeneration.

Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress.

Partitioning-defective protein 6 regulates insulin-dependent glycogen synthesis via atypical protein kinase C.

The atypical isoforms of protein kinase C (aPKCs) play an important role in insulin signaling and are involved in insulin-stimulated glucose uptake in different cell systems. On the other hand, aPKCs also are able to negatively regulate important proteins for insulin signaling, like phosphatidylinositol 3-kinase and protein kinase B/Akt. To find aPKC-interacting proteins that may promote positive or negative activities of aPKCs, a yeast two-hybrid screen was performed. Partitioning-defective protein 6 (Par6) was detected in human cDNA libraries of different adult insulin-sensitive tissues. Although Par6 is known as an aPKC-interacting protein during development, no role for Par6 in insulin signaling has been reported so far. We therefore studied the effects of Par6 overexpression in C2C12 murine myoblasts. In these cells, Par6 associated constitutively with endogenous aPKCs, and the expression level as well as the activity of aPKCs were increased. Insulin-dependent association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate 1 was hampered and the phosphorylation of Akt/glycogen synthase kinase-3alpha/beta was significantly impaired after stimulation with insulin or with platelet-derived growth factor. Consequently, insulin-dependent glycogen synthesis was down-regulated (1.44 vs. 2.24 fold, P < 0.01). We therefore suggest that Par6 acts as a negative regulator of the insulin signal.

The Schistosoma mansoni Src kinase TK3 is expressed in the gonads and likely involved in cytoskeletal organization.

Cytoplasmic protein tyrosine kinases of the Src family play a pivotal role in the regulation of cellular processes including proliferation and differentiation. Among other functions, Src kinases are involved in regulating the cell architecture. In an approach to identify protein tyrosine kinases from the medically important parasite Schistosoma mansoni, we isolated the TK3 gene by degenerate primer PCR and cDNA library screening. Sequencing of the complete cDNA and data-base analyses indicated that TK3 is a Src family kinase. Its predicted size of 71 kDa was confirmed by Western blot analysis. Southern blot analysis showed that TK3 is a single-copy gene, and Northern blot and RT-PCR experiments indicated its expression in both sexes and throughout development. Localization studies by in situ hybridization and immunolocalization revealed that TK3 is predominantly expressed in the reproductive organs such as the testes of the male and the ovary as well as the vitellarium of the female. Its enzymatic activity was confirmed by functional analyses. In transient transfection experiments with HEK293 cells, TK3 phosphorylated the well-known Src-kinase substrate p130 Cas, an intracellular scaffolding protein. Yeast two-hybrid screenings in a heterologous invertebrate system identified dAbi, vinculin and tubulin as binding partners, representing molecules that fulfill functions in the cell architecture of many organisms. These findings suggest that TK3 may play a role in signal transduction pathways organizing the cytoskeleton in the gonads of schistosomes.

HSP70-controlled GFP expression in transiently transformed schistosomes.

Among the parasitic helminths schistosomes are of high medical and economic importance. Despite of the world-wide relevance of this parasite, very little is known about the cellular mechanisms controlling its development and concerning the host-parasite interaction. Within the last decade a great effort has been made in this blood fluke to identify genes which play important roles during these processes. However, molecular analysis was limited by the fact, that neither function nor regulation of candidate genes could be investigated in this organism due to the lack of transformation protocols. Here, we present the strategy of ballistic gene transfer to introduce and characterize transgenes in different schistosome life stages. As a transformation vector, the heat shock protein 70 (hsp70) gene promoter and terminator from Schistosoma mansoni were cloned and fused to the green fluorescent protein (GFP) reporter gene. In a first attempt, the hsp70--GFP vector was successfully tested in a eukaryotic cell line. Thereafter, adult male schistosomes and sporocysts were transformed with this vector, and GFP expression was demonstrated using molecular and microscopical methods. PCR, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses confirmed the presence, transcription and translation of the transgene in adults. Confocal laser scanning microscopy revealed GFP-activity at various sites along the surface of the worms after hs induction and within sporocysts. These results suggest diverse roles for hsp70 during the development of schistosomes. Furthermore, the results demonstrate the feasibility of this method and open the perspective to analyze a variety of molecular functions in schistosomes.

Characterisation of the cysteine protease ER60 in transgenic Schistosoma mansoni larvae.

Proteinases have been found to play important roles in parasites. They are involved in developmental processes and facilitate invasion of host tissues as well as the digestion of host molecules for nutrition. The cysteine protease ER60 from Schistosoma mansoni, originally characterised in adults to be expressed in excretory organs, was analysed in larval stages. Transcripts were found in miracidia, in vitro generated mother sporocysts and cercariae. After cloning the promoter and terminator of the ER60 gene, a transformation vector was constructed containing the green fluorescent protein reporter gene flanked by the regulatory elements. The ER60-green fluorescent protein vector was used for transfection experiments of COS-7 cells demonstrating the functionality of the promoter in the heterologous system. To analyse the expression pattern of ER60-green fluorescent protein in larval S. mansoni, in vitro generated mother sporocysts were transformed by particle bombardment, a method which allows gene transfer into schistosomes. Molecular analyses demonstrated transcription and translation of the transgene. Furthermore, confocal laser scanning microscopy revealed ER60-induced green fluorescent protein fluorescence within the larvae. Inside primary sporocysts, tissue-specific activity was localised in the gland cells, protonephridia and several cytons. These results suggest that ER60 is expressed in the ES system of larvae and, amongst other functions, may play a role in penetration and migration processes.

Proteolytic processing of the protein tyrosine phosphatase α extracellular domain is mediated by ADAM17/TACE.

The receptor protein tyrosine phosphatase alpha (PTPα) is involved in the regulation of tyrosine kinases like the Src kinase and the insulin receptor. As with other PTPs, its function is determined by alternative splicing, dimerisation, phosphorylation and proteolytical processing. PTPα is cleaved by calpain in its intracellular domain, which decreases its potential to dephosphorylate Src kinase. Here, we demonstrate that PTPα is also processed in the extracellular domain. Extracellular processing was exclusively found for a splice variant containing an extra nine amino acid insert three residues amino-terminal from the transmembrane domain. Processing was sensitive to the metalloprotease-inhibitor Batimastat, and CHO-M2 cells lacking a disintegrin and metalloproteinase 17 (ADAM17; tumor-necrosis-factor α converting enzyme) activity were not able to cleave PTPα. After transient overexpression of ADAM17 and PTPα in these cells, processing was restored, proving that ADAM17 is involved in this process. Further characterization of the consequences of processing revealed that dephosphorylation of the insulin receptor or activation of Src was not affected but focus formation was reduced. We conclude that extracellular proteolytic processing is a novel mechanism for PTPα regulation.

The Drosophila Crumbs signal peptide is unusually long and is a substrate for signal peptide peptidase.

N-terminal signal sequences mediate nascent protein targeting to and protein insertion into the membrane of the endoplasmic reticulum. They are typically 15-30 amino acid residues long with a core hydrophobic region flanked by an N-terminal (n-) and a C-terminal region. Following cleavage by signal peptidase, some of the resulting signal peptides are further processed by signal peptide peptidase (SPP) and fragments are liberated into the cytosol. Such fragments can have independent, post-targeting functions affecting diverse cellular processes. We show that Drosophila melanogaster Crumbs, a transmembrane protein controlling cell polarity and morphogenesis, is synthesized with an 83 residues-long signal sequence. To our knowledge, this is currently the longest signal sequence described for an eukaryotic protein. The unusual length is caused by an extended n-region, but the extension does neither affect protein targeting nor signal sequence cleavage. The signal sequence is cleaved off and the resulting signal peptide, SP(Crb), is proteolytically processed by SPP, thus representing the first substrate described for the Drosophila enzyme. We further show that signal peptide fragments can be degraded by the proteasome. Expression of transgenes encoding tagged variants of Crumbs in Drosophila embryos suggests that the signal peptide is short-lived in vivo. Our findings support a model suggesting that besides generating fragments with post-targeting functions, SPP-mediated processing is the first step in the degradation of signal peptides.

The signal peptide of the mouse mammary tumor virus Rem protein is released from the endoplasmic reticulum membrane and accumulates in nucleoli.

N-terminal signal sequences mediate endoplasmic reticulum (ER) targeting and insertion of nascent secretory and membrane proteins and are, in most cases, cleaved off by signal peptidase. The mouse mammary tumor virus envelope protein and its alternative splice variant Rem have an unusually long signal sequence, which contains a nuclear localization signal. Although the envelope protein is targeted to the ER, inserted, and glycosylated, Rem has been described as a nuclear protein. Rem as well as a truncated version identical to the cleaved signal sequence have been shown to function as nuclear export factors for intron-containing transcripts. Using transiently transfected cells, we found that Rem is targeted to the ER, where the C-terminal portion is translocated and glycosylated. The signal sequence is cleaved off and accumulates in nucleoli. In a cell-free in vitro system, the generation of the Rem signal peptide depends on the presence of microsomal membranes. In vitro and in cells, the signal peptide initially accumulates in the membrane and is subsequently released into the cytosol. This release does not depend on processing by signal peptide peptidase, an intramembrane cleaving protease that can mediate the liberation of signal peptide fragments from the ER membrane. Our study suggests a novel pathway by which a signal peptide can be released from the ER membrane to fulfill a post-targeting function in a different compartment.

Extracellular domain splice variants of a transforming protein tyrosine phosphatase alpha mutant differentially activate Src-kinase dependent focus formation.

The extracellular domains of receptor-type protein-tyrosine phosphatases (PTPs) contain a diverse range of protein modules like fibronectin- or immunoglobulin-like structures. These are frequently expressed in a tissue- and development specific manner as splice variants. The extracellular domain of PTPalpha is rather short and heavily glycosylated. Two splice variants are known, which it differs by an exon encoding nine amino acids within the extracellular domain. We have analyzed the expression pattern of both variants and found that the smaller form is ubiquitously expressed while the larger form was found at an increased level only in brain, some skeletal muscle and differentiating cells like granule neurons, adipocytes and myotubes. The phosphatase activity of both forms was similar when tested in vitro using para-nitrophenylphosphate as a substrate and in a transient expression system with the substrates c-Fyn or c-Src. In a quantitative focus formation assay the capability of the larger form to activate Src-dependent focus formation in intact cells was increased more than twofold whereas the capability to dephosphorylate the insulin receptor in a BHK cell system was similar. We conclude that the two splice variants of PTPalpha are expressed differentially and regulate c-Src activity in different ways.

The protein tyrosine phosphatase alpha modifies insulin secretion in INS-1E cells.

Increasing evidence indicates a role of insulin signalling for insulin secretion from the pancreatic beta-cells. Therefore, regulators of insulin signalling, like protein tyrosine phosphatases, could also have an impact on insulin secretion. Here, we investigated a possible role of the negative regulator protein tyrosine phosphatase alpha (PTP alpha) for insulin secretion. RT-PCR analysis confirmed that both splice variants of the extracellular domain of PTP alpha that vary by an insert of 9 amino acids are expressed in human islets and insulinoma cells (INS-1E, RIN1046-38). Overexpression of the wild type PTP alpha splice variant containing the 9 amino acids reduced insulin secretion, as did a mutant form unable to bind Grb2 (Tyr798Phe). By contrast, overexpression of a phosphatase inactive mutant improved insulin secretion. These data reveal a functional relevance of PTP alpha for insulin secretion.

Transplantation of in vitro-generated Schistosoma mansoni mother sporocysts into Biomphalaria glabrata.

Specific studies on schistosome gene functions require both access to the parasite stages, preferably the larvae, and to complete the life cycle. In the present study, we investigated whether short-term in vitro cultivation of sporocysts and surgical transplantation into snails could be combined to produce cercariae. Miracidia were maintained in vitro in the presence of Biomphalaria glabrata embryonic (Bge) cells or, alternatively, in Bge-cell-conditioned medium. The transformation of miracidia to mother sporocysts was observed in both cases. Two day-old sporocysts were transplanted into the cephalopedal sinus of recipient snails. Transplantation efficiencies varied between 16% and 43%, depending on the culture of the sporocysts in terms of the number of cercariae producing snails. Cercariae recovered from these snails were used to successfully infect hamsters, demonstrating that short term in vitro-generated sporocysts undergo normal cercariogenesis following transplantation. This combination of in vitro cultivation and transplantation may be useful for novel experimental approaches to investigate the genes involved in larval development or host-parasite molecular interactions.