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Fig. 5. Gene constructs. (A) Schematic diagram of genomic structure of Ce-imp genes. (B) Plasmids used for dsRNAi experiments by feeding. Double-T7 vector L4440 was used for subcloning of cDNA of Caenorhabditis elegans genes: Ce-imp-2; Ce-lrp-1. To confirm the dsRNAi result, we performed experiments using different subfragments of full-length Ce-imp-2 cDNA cloned into L4440 vector.

Fig. 6. Schematic presentation of Ce-LRP1 protein and Ce-lrp-1 L-ICD construct, corresponding to intracellular domain of Ce-LRP1. Putative adaptor binding sites (FxNPxY and PxxPxxP) are shown (red arrows). Ce-lrp-1 L-ICD construction was used for Ce-imp-2 RNAi ISC rescue experiments in C. elegans. Mammalian lipoprotein receptor-related proteins (LRPs) integrate endocytosis and signal transduction. The large extracellular domain of megalin is required for binding to diverse ligands from proteases to lipids. The intracellular part of megalin contains multiple motifs essential for interactions with different adaptor molecules, e.g., disabled-2 (Dab2), megalin-binding protein (MegBP), ankyrin repeat-containing protein (ANKRA) associated with transcription regulator skeletal muscle- and kidney-enriched inositol phosphatase (SKIP), mitogen-activated protein kinase (MAPK), and membrane-associated guanylate kinase-1 (MAGI-1) kinases and kinase interacting protein c-Jun amino-terminal kinase (JNK)-interacting protein (JIP) (1-4). Ce-LRP-1 has a similar structure with a motif in the intracellular domain for binding with Ce-DAB-1 (FxNxPY) (5) and a proline-rich motif (PxxPxxP) for potential binding with other adaptor proteins.

1. Gotthardt, M., Trommsdorff, M., Nevitt, M. F., Shelton, J., Richardson, J. A., Stockinger, W., Nimpf, J. & Herz, J. (2000) J. Biol. Chem. 275, 25616-25624.

2. Rader, K., Orlando, R. A., Lou, X. & Farquhar, M. G. (2000) J. Am. Soc. Nephrol. 11, 2167-2178.

3. Petersen, H. H., Hilpert, J., Militz, D., Zandler, V., Jacobsen, C., Roebroek, A. J. & Willnow, T. E. (2003) J. Cell Sci. 116, 453-461.

4. May, P., Bock, H. H. & Herz, J. (2003) Sci. STKE. 2003, E12.

5. Kamikura, D. M. & Cooper, J. A. (2003) Genes Dev. 17, 2798-2811.

Fig. 7. Similarity of Ce-IMP- to other members of IMPAS and presenilin families of proteins. Shown is multiple alignment (alibee) of most conserved domains of presenilin and IMPASes from distant taxons. Presenilins: PSEN1,2, Homo sapiens; SEL-12, C. elegans. IMPASes: Ce-IMP-1-3, C. elegans; hIMP1-5, H. sapiens; Dr.-IMP1,2, Drosophila melanogaster; S.pomb-IMP, Schizosaccharomyces pombe; Ar.-IMP, Arabidopsis thaliana AAC34490; related proteins from Arhaea-Arch.fulg., Archaeoglobus fulgidus, AAB89302. Groups of similar aa: 1-D,N; 2-E,Q; 3-S,T; 4-K,R; 5-F,Y,W; 6-L,I,V,M (Blosum 62 scoring matrix). Amino acids of 100%, 80%, and 60% homology are marked in black, dark gray, and light gray, respectively (genedoc editing program). The total protein length is indicated in brackets. Arrowheads indicate most conserved aspartate residues and PAL-motif. The percent (%) of identity and similarity between members of C. elegans IMP proteins are: Ce-IMP-1/ Ce-IMP-2 (17/31); Ce-IMP-2/ Ce-IMP-3 (14/31); Ce-IMP-1/ Ce-IMP-3 (15/30); and for the most conserved domains (corresponding to amino acid 269-444 of Ce-IMP-2): Ce-IMP-1/ Ce-IMP-2 (28/47); Ce-IMP-2/ Ce-IMP-3 (20/48); Ce-IMP-1/ Ce-IMP-3 (14/37).

Fig. 8. Expression of human IMP1 in mammalian cells and S3-cleavage of Notch1 receptor. (A) Transiently or stably transfected hIMP1 in human embryonic kidney (HEK293), Chinese hamster ovary (CHO), mouse fibroblasts, and rat PC12 (Upper) and endogenous hIMPs in HEK293, human neuroglyoma (H4) or PC12 cells (Lower Left) are detected by Western blot as two major 42- to 48-kDa and 95- to 98-kDa fractions and, occasionally, as an » 135- to 145-kDa fraction . The 42- to 48-kDa fraction of predicted glycosylated hIMP1 holoprotein (1) is increased in follow-up procedures or freezing–defreezing or heating of the cell lysates. The IMP1 molecules tagged to two different epitopes (c-myc and V5) are coimmunoprecipitated, suggesting that the fraction of 95-98 kDa likely represents homodimeric isoforms resistant to SDS denaturing conditions (Lower Right). Other high molecular weight fractions may represent more complex aggregates of hIMP1 proteins. The C. elegans Ce-IMP-2 transiently expressed in human HEK293 cells was detected only as a predicted 50- to 55-kDa monomer fraction. The antibodies against c-myc or V5 epitopes fused to the IMPs were used in Western blot detection. (B, C, and D) Overexpression of wild-type hIMP1 does not facilitate cleavage of Notch1 as detected by pulse-chase analysis. HEK293 cells were cotransfected with the constructs: (i) NotchD E c-myc tag and (ii) hIMP1 wt is fused to c-myc or V5 tags in pcDNA4/myc-HisB and pcDNA6/V5-HisA vectors or PS1 D385A or mock (pcLacZ). NICD is a predicted derivate of NotchD S3 cleavage. In follow-up pulse-labeling procedures, proteins were immunoprecipitated with anti-c-myc antibodies and subjected to electrophoresis and autoradiography. As described, the NICD product of S3 cleavage is clearly detected in 60 min of pulse-chase in HEK293 cells expressing membrane-tethering NotchD E (A and B). The coexpression of NotchD E with loss-of-function PS1 D385A mutant has dominant-negative effect suppressing S3 activity of endogenous PS. The hIMP1 wt does not increase, but reduces efficiency of S3 cleavage (see also Fig. 2). (D) Histogram showing quantitation of NotchD E S3-cleavage product. NotchD E S3-cleavage is not increased, but reduced in cells overexpressing hIMP1. Autoradiographic films were scanned, and images were analyzed using quantity one 4.3.0 image analysis software (Bio-Rad). The signal volume of NICD was normalized against the total Notch (NotchD E plus NICD) for each line. Bar graphs represent mean value and SEs for mock (n = 3), PS1D385A (n = 3), and hIMP1 (n = 4).

Note. Nyborg et al. (2) have recently also indicated coimunoprecipitation of hIMP1/SPP molecules using other constructs and cell lysis conditions.

1. Moliaka, Y. K., Grigorenko, A., Madera, D. & Rogaev, E. I. (2004) FEBS Lett. 557, 185-192.

2. Nyborg, A. C., Kornilova, A. Y., Jansen, K., Ladd, T. B., Wolfe, M. S. & Golde, T. E. (2004) J. Biol. Chem. 279, 15153-15160.

Fig. 9. Ce-imp-2 RNAi phenotypes on different stages (performed on N2 strain). Nomarski optic longitudinal views of C. elegans phenotypes. Shown are early (A) and late (B) dead embryos. (C) Dead L₁ worm soon after hatching. ISC during molting stages: (D) L₁-L₂, (E) L₂-L₃, (F) L₃-L₄, (J) L₄-adult. (H) ISC phenotype produced by suppression effect of injected transgenic genomic Ce-imp-2 construct. Expression of injected Ce-imp-2 genomic transgenic DNA occasionally produced phenotypes similar to Ce-imp-2 RNAi defects. This effect may be explained by a dominant-negative effect of the transgene constructs or a cosuppression phenomenon phenocopying the loss-of-function phenotype described for multiple copy transgenes in C. elegans and other organisms (1). (I) Progeny hatched inside N2 worm with ISC. (A-I) Black arrows indicate ISC. (J) Exploded body (Rup) phenotype is occasionally detected in N2 Ce-imp-2 RNAi worms (indicated with black arrows).

1. Ketting, R. F. & Plasterk, R. H. (2000) Nature 404, 296-298.

Fig. 10. Hypothetical model of Ce-imp-2/IMP1 and cholesterol-lipoprotein receptor (Ch-LR)-dependent pathway in development. C. elegans cannot synthesize cholesterol and depends on minimal amounts of exogenous cholesterol (1). It seems, therefore, that cholesterol plays a minimal role in membrane structures in C. elegans. The role of cholesterol or cholesterol-derived sterol as the most upstream molecule in signal transduction and Ch-LR-dependent development is thus suggested . We identified Ce-imp-2 as one of these putative components, presumably acting upstream of Ce-lrp-1. Interestingly, in retrospect, cholesterol-dependent Hedgehog signaling required for development and neurogenesis has been linked to megalin function in vertebrates. Embryos deficient in megalin and Sonic hedgehog have similar development abnormalities, including a holoprosencephalic phenotype in mouse. Megalin was suggested as an endocytic receptor for Sonic hedgehog (Shh)-secreted protein (2, 3). There are at least 10 genes in C. elegans encoding domains homologous to mammalian hedgehog proteins (4), but their function is not yet known. Mammalian Dab2 also interacts with clathrin adaptor proteins (5). Therefore, other proteins involved in exo- and endocytosis (e.g., in clathrin-mediated endocytosis steps) might potentially interact with the Ce-imp-2-regulated pathway. Conservation of the cholesterol-dependent pathway in C. elegans and mammals is postulated. Abnormalities in regulatory elements of the pathway lead to specific phenotypes of molting defects in C. elegans (ISC). This C. elegans ISC phenotype has been described for defects in daf-9 (upstream regulator of daf-12 nuclear receptor), which is the ortholog of CYP17, CYP21-critical enzymes for synthesis of sterols in mammals; Ce-lrp-1 membrane receptor gene; Ce-dab-1 gene, encoding the intracellular adaptor protein Disabled. Human or mouse orthologs of these genes appear to be also involved in cholesterol–regulated development or signaling. Nuclear receptors are transcriptional factors in Metazoa. C. elegans nhr -25 (ortholog of mammalian FTZ-F1) is required for hypodermal and somatic gonad development. Defects in nhr-23,nhr-25 include ISC. Mammalian LRH-1 (FTZ-F1 like) nuclear receptor is involved in cholesterol homeostasis (6). The model implicates the Ce-imp-2/IMP1 intramembrane protein in Ch-LR homeostasis or signaling. Orthologous genes in C. elegans are shown in italics.

1. Kurzchalia, T. V. & Ward, S. (2003) Nat. Cell Biol. 5, 684-688.

2. Farese, R. V., Jr., & Herz, J. (1998) Trends Genet. 14, 115-120.

3. McCarthy, R. A. & Argraves, W. S. (2003) J. Cell Sci. 116, 955-960.

4. Aspock, G., Kagoshima, H., Niklaus, G. & Burglin, T. R. (1999) Genome Res. 9, 909-923.

5. Morris, S. M., Tallquist, M. D., Rock, C. O. & Cooper, J. A. (2002) EMBO J. 21, 1555-1564.

6. Nitta, M., Ku, S., Brown, C., Okamoto, A. Y. & Shan, B. (1999) Proc. Natl. Acad. Sci. USA 96, 6660-6665.