Ribosome-inactivating proteins (RIPs) from angiosperms are rRNA N-glycosidases that have been proposed as defence proteins against virus and fungi. They have been classified as type 1 RIPs, consisting of single-chain proteins, and type 2 RIPs, consisting of an A chain with RIP properties covalently linked to a B chain with lectin properties. In this work we have carried out a broad search of RIP sequence data banks from angiosperms in order to study their main structural characteristics and phylogenetic evolution. The comparison of the sequences revealed the presence, outside of the active site, of a novel structure that might be involved in the internal protein dynamics linked to enzyme catalysis. Also the B-chains presented another conserved structure that might function either supporting the beta-trefoil structure or in the communication between both sugar-binding sites. A systematic phylogenetic analysis of RIP sequences revealed that the most primitive type 1 RIPs were similar to that of the actual monocots (Poaceae and Asparagaceae). The primitive RIPs evolved to the dicot type 1 related RIPs (like those from Caryophyllales, Lamiales and Euphorbiales). The gene of a type 1 RIP related with the actual Euphorbiaceae type 1 RIPs fused with a double beta trefoil lectin gene similar to the actual Cucurbitaceae lectins to generate the type 2 RIPs and finally this gene underwent deletions rendering either type 1 RIPs (like those from Cucurbitaceae, Rosaceae and Iridaceae) or lectins without A chain (like those from Adoxaceae).

Ebulin l is a type-II ribosome-inactivating protein (RIP) isolated from the leaves of Sambucus ebulus L. As with other type-II RIP, ebulin is a disulfide-linked heterodimer composed of a toxic A chain and a galactoside-specific lectin B chain. A normal level of ribosome-inactivating N-glycosidase activity, characteristic of the A chain of type-II RIP, has been demonstrated for ebulin l. However, ebulin is considered a nontoxic type-II RIP due to a reduced cytotoxicity on whole cells and animals as compared with other toxic type-II RIP like ricin. The molecular cloning, amino acid sequence, and the crystal structure of ebulin l are presented and compared with ricin. Ebulin l is shown to bind an A-chain substrate analogue, pteroic acid, in the same manner as ricin. The galactoside-binding ability of ebulin l is demonstrated crystallographically with a complex of the B chain with galactose and with lactose. The negligible cytotoxicity of ebulin l is apparently due to a reduced affinity for galactosides. An altered mode of galactoside binding in the 2gamma subdomain of the lectin B chain primarily causes the reduced affinity.

The bark of Sambucus nigra L. contains a non-toxic novel type 2 ribosome-inactivating protein that we named nigrin b. In vitro, nigrin b strongly inhibited mammalian protein synthesis but did not affect plant nor bacterial protein synthesis. The protein (M(r) 58,000) contains two subunits, A (M(r) 26,000) and B (M(r) 32,000); linked by disulphide bridge(s). Nigrin b was found to be an rRNA N-glycosidase of the rRNA of intact mammalian ribosomes and shares a very good N-terminal amino-acid sequence homology with the anti-HIV-1 proteins TAP 29 and trichosanthin.

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolincontaining fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment. INTRODUCTIONAcidic FGF (aFGF or FGF-1) belongs to the large FGF family of growth factors that play important roles in cell proliferation and differentiation (Burgess and Maciag, 1989;Crumley et al., 1991;Basilico and Moscatelli, 1992). The growth factors bind to transmembrane receptors with a cytoplasmic split tyrosine kinase domain. There are four identified FGF receptors (FGFRs) and a number of splicing variants (Johnson et al., 1990(Johnson et al., , 1991Hou et al., 1991;Partanen et al., 1991;Chellaiah et al., 1994). Different FGFs bind with different strengths to the different variants of the receptors. In addition, FGFs bind to cell surface heparans (Burgess and Maciag, 1989) as well as to a cysteine-rich binding protein of unknown function (Burrus et al., 1992;Gonatas et al., 1995).aFGF induces a strong mitogenic response in some cells, whereas in other cells it induces differentiation (Burgess and Maciag, 1989). It is not known what mechanism determines which response will be induced in each case. Upon binding of the growth factor to receptors, the tyrosine kinase is activated, apparently as a result of dimerization of the receptors by the bound growth factor (DiGabriele et al., 1998). Subsequently, a number of downstream signaling molecules are activated, such as phospholipase C␥ and MAPK (Mason, 1994). There is increasing evidence that externally added aFGF also acts intracellularly and that upon binding to the receptor the growth factor is able to penetrate cellular membranes and enter the cytosol and the nucleus (Imamura et al., 1990(Imamura et al., , 1994Zhan et al., 1992Zhan et al., , 1993Wiedlocha et al., 1994;Muñ oz et al., 1997;Klingenberg et al., 1998).After binding to FGFR, aFGF is internalized by endocytosis (Muñ oz et al., 1997). It has so far not been...

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