An important negative control mechanism in the signaling of epidermal growth factor (EGF) is the endocytosis and subsequent degradation of activated EGF receptors. Eps15 and its related partner Eps15R play a key role in clathrin-mediated endocytosis of transmembrane receptors. Upon EGF stimulation of the cell, Eps15 becomes both phosphorylated on tyrosine residues and monoubiquitinated. Although tyrosine phosphorylation of Eps15 has been implicated in EGF receptor internalization, the function of Eps15 ubiquitination is not known. Using a mutational approach, we have found that the second ubiquitin-interacting motif (UIM) of Eps15 and Eps15R is essential for their ubiquitination. This UIM partially overlaps with the recently characterized nuclear export signal in Eps15. We show that these two overlapping motifs have different structural requirements with respect to nuclear export signal versus ubiquitination signal activity. Our data demonstrate that the UIM does not contain the ubiquitin acceptor site but functions as a recruitment site for the ubiquitination machinery leading to the monoubiquitination of both Eps15 and Eps15R.

Three distinct cDNAs encoding the preproadipokinetic hormones I, II, and III (prepro-AKH I, II, and III), respectively, of Locusta migratoria have been isolated and sequenced. The three L. migratoria AKH precursors have an overall architecture similar to that of other precursors of the AKH/red pigment-concentrating hormone (RPCH) family identified so far. The AKH I and II precursors of L. migratoria are highly homologous to the Schistocerca gregaria and Schistocerca nitans AKH precursors. Although the L. migratoria AKH III precursor appears to be the least homologous to the Manduca sexta, Drosophila melanogaster, and Carcinus maenas AKH/RPCH precursors, we favor the opinion that the L. migratoria AKH III precursor is evolutionary more related to the M. sexta, D. melanogaster, and C. maenas AKH/RPCH precursors than to the AKH I and II precursors of S. gregaria, S. nitans, or L. migratoria. In situ hybridization showed signals for the different AKH mRNAs to be co-localized in cell bodies of the glandular lobes of the corpora cardiaca. Northern blot analysis revealed the presence of single mRNA species encoding the AKH I precursor (ϳ570 bases), AKH II precursor (ϳ600 bases), and AKH III precursor (ϳ670 bases), respectively. Interestingly, flight activity increased steady-state levels of the AKH I and II mRNAs (ϳ2.0 times each) and the AKH III mRNA (ϳ4.2 times) in the corpora cardiaca.Three peptide hormones with hyperlipemic activity, the adipokinetic hormones I, II and III (AKH 1 I, II and III; see Table I) (1-3), are synthesized by the glandular neurosecretory cells of the corpora cardiaca (CC) of the migratory locust, Locusta migratoria. These peptides are members of a large family of structurally related but functionally diverse peptides (the AKH/RPCH family) (4). In the adult locust, the AKHs I and II are released into the hemolymph during flight and are involved in the mobilization of lipid and carbohydrate from the fat body to serve as energy substrates for the flight muscles (4 -7). Data on the release and functioning of AKH III are lacking so far. Isolation and characterization of CC peptides revealed that two other locust species, Schistocerca gregaria and Schistocerca nitans, each contain two AKHs that are mutually identical (1, 8), whereas Manduca sexta and Drosophila melanogaster each contain only one AKH (9, 10) (see Table I).Molecular biological studies have resulted in the characterization of the structure of the AKH/RPCH precursors (a signal peptide, AKH/RPCH, a Gly-(Lys/Arg)-Arg sequence, and an AKH/RPCH-associated peptide (AAP/RAP), in this order) of S. gregaria, S. nitans, M. sexta, D. melanogaster,.The biosynthesis of the AKHs in S. gregaria has been elucidated in detail by O'Shea and co-workers (13, 18 -21). The signal peptide is co-translationally removed from prepro-AKH, generating pro-AKH. Next, proteolytic processing, which is preceded by dimerization of two pro-AKHs (I/I, I/II, or II/II) via their single COOH-terminal Cys residues, gives rise to two AKHs (I and/or II) and one homo-or hete...

SUMMARYWe present a modified diaminobenzidine (DAB) photoconversion method that enables staining of internalized DiI-labeled lipoproteins without the apparent punctate background staining that was observed with the original DAB photoconversion method. This is illustrated by the localization of DiI-labeled insect lipoproteins in natural recipient cells that internalize these lipoproteins by receptor-mediated endocytosis. Exposure to DiI-excitation light of cells that had been incubated with DiI-labeled lipoproteins yielded a light-and electron-dense DAB reaction product. In addition to the expected staining, an apparent punctate background staining of vesicular structures hindered proper identification of DiI-containing vesicles because these background-stained vesicles were indistinguishable from putative late endosomal and lysosomal structures at the electron microscopic level. This background staining was completely abrogated by inhibition of peroxisomal catalase with aminotriazole. The conversion of DAB by the emitted light of DiI was not affected by aminotriazole. We conclude that specific staining of DiI-labeled intracellular structures can be achieved with the modified DAB photoconversion method reported here. (J Histochem Cytochem 46:1085-1089, 1998) Maranto (1982) reported that illumination of Lucifer Yellow in the presence of diaminobenzidine (DAB) resulted in the formation of a dark-stained DAB reaction product that was visible at light and electron microscopic levels. This method was originally developed for neuronal mapping and has hitherto been applied mainly for microscopic tracing of fluorescently labeled neurons (Sandell and Masland 1988;Von Bartheld et al. 1990;Lübke 1993). In these studies, DAB photoconversion was found to be a general phenomenon, because illumination of different fluorescent dyes resulted in the same apparent DAB reaction product.We attempted to apply DAB photoconversion for visualization of DiI-labeled insect lipoproteins internalized by the natural recipient tissue, the fat body. In preliminary studies we noted that the original DAB photoconversion technique cannot be used for this purpose, because of a prominent punctate background staining that seriously hindered identification of DiI-containing structures. Similar staining and its implications have been reported previously for DAB photoconversion by DiI-labeled mammalian very low-density lipoproteins and low-density lipoproteins in macrophages (Tabas et al. 1990). In this report we describe a modified DAB photoconversion method that completely abrogates the background staining, whereas the actual photoconversion was unaffected. With this method, DiI-labeled lipoproteins could be visualized at the light microscopic level and in detail at the electron microscopic level. Materials and Methods AnimalsMigratory locusts, Locusta migratoria, were reared under crowded conditions as described previously (Van der Horst Correspondence and present address: N. P. Dantuma, Microbiology and Tumor

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