The eukaryotic proteasome is a barrel-shaped protease complex made up of four seven-membered rings of which the outer and inner rings may contain up to seven different ␣-and ␤-type subunits, respectively. The assembly of the eukaryotic proteasome is not well understood. We cloned the cDNA for HsC8, which is one of the seven known human ␣-type subunits, and produced the protein in Escherichia coli. Recombinant HsC8 protein forms a complex of about 540 kDa consisting of double ringlike structures, each ring containing seven subunits. Such a structure has not earlier been reported for any eukaryotic proteasome subunit, but is similar to the complex formed by the recombinant ␣-subunit of the archaebacterium Thermoplasma acidophilum (Zwickl, P., Kleinz, J., and Baumeister, W. (1994) Nat. Struct. Biol. 1, 765-770). The ability of HsC8 to form ␣-rings suggests that these complexes may play an important role in the initiation of proteasome assembly in eukaryotes. To test this, we used two human ␤-type subunits, HsBPROS26 and HsDelta. Both these ␤-type subunits, either in the proprotein or in the mature form, exist in monomers up to tetramers. In contrast to the ␣-and ␤-subunit of T. acidophilum, coexpression of the human ␤-type subunits with HsC8 does not result in the formation of proteasome-like particles, which would be in agreement with the notion that proteasome assembly in eukaryotes is much more complex than in archaebacteria.

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alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli. The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.

External application of auxin and cytokinin is required for the formation of flower buds on thin-layer tissue explants of Nicotiana tabacum cv Samsun. Interaction between both plant growth regulators during this regenerative process has been demonstrated with respect to speed of flower bud initiation and the number of flower buds formed. Separation in time of the hormone application during culture revealed that the cytokinin benzyladenine plays a key role in flower bud initiation whereas auxin (indoleacetic acid) stimulates in particular the differentiation of flower buds. The uptake of each hormone was proportional to the concentration supplied in the medium, and the uptake of either hormone appeared independently of the presence of the other. Metabolism studies showed the conversion of indoleacetic acid by the tissue to at least 13 metabolites after 24 h of culture. In addition, indoleacetic acid metabolism was demonstrated not to be influenced by the uptake and metabolism of benzyladenine. Taken together the results indicate that the interaction of auxin and cytokinin with respect to in vitro flower bud formation is indirect, i.e. does not take place at the level of hormone uptake or metabolism but at some step in the cascade of processes they initiate.Flowering is a unique developmental event in the life cycle of a higher plant and results from a redetermination of the vegetative shoot meristem. In contrast to the reiterative leaf- Similarly, examination of in vitro cultures ofthin cell layers consisting of epidermis and subepidermal cortex from N. tabacum cv Samsun reveals determination by the formation of subepidermal meristems induced by hormones that, depending upon the type of tissue, may develop into either vegetative shoots or floral buds (2,(19)(20)(21)25). Our studies are focused on floral bud formation on thin-layer tobacco pedicel explants that regenerate only flower buds directly from epidermal and/or subepidermal cells without intermediate callus formation (25).In this in vitro system, regeneration of flower buds can be studied without interference by external signals from other plant parts and the environmental factors can be controlled. Two hormones, auxin and cytokinin, are required for the induction of flower bud initiation on tobacco thin-layer explants. The (interactive) effects of these hormones are expressed in three ways: (a) the number of flower buds formed per explant, (b) the speed of bud formation, and (c) the distribution of flower buds over the explant surface (2,12,14,23). Cytokinin determines in particular whether flower buds are formed, whereas auxin mainly determines the position of buds on the explants. In addition, it was shown that both hormones were rapidly metabolized and that the level of flower bud formation is regulated by the concentrations of the free, nonmetabolized hormones (14, 23).The aim of this study was threefold: (a) to compare the effect of the natural auxins (IAA)3 and IBA and the synthetic NAA with respect to in vitro induction of flower buds; (b) ...

The barrel-shaped 20S proteasome is one of the two components of a larger 26S particle, the multicatalytic 2000-kDa protease complex. The proteolytic sites are located in the inner chamber of the 20S particle and are only accessible via narrow entrances. This paper reviews the current knowledge concerning proteasome formation, proteolytic activities, structural aspects and assembly. Eukaryotic proteasomes are made up by four rings each of which contains seven different subunits occurring at fixed positions. While the outer rings contain alpha-type subunits, the inner ones comprise beta-type subunits. The current assembly model for eukaryotic 20S proteasomes is based upon the detection of 13S and 16S intermediates, respectively, in addition to previous findings with archaebacterial and eubacterial proteasome assembly. The available data suggest a cooperative assembly of the alpha-type and beta-type subunits into half proteasome-like complexes followed by dimerization into proteasomes. During or after dimerization of half proteasomes, the beta-type subunits are processed. The prosequence of the beta-type subunits is essential for the assembly proves and prevents protease activity of immature proteasomes.