- Email: [email protected]
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12 Cruz, A.K., Coburn, C.M. and Beverley, S.M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7170–7174 13 Titus, R.G. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10267–10271 14 Gueiros-Filho, F.J. and Beverley, S.M. (1996) Mol. Cell. Biol. 16, 5655–5663 15 Nare, B., Hardy, L.W. and Beverley, S.M. (1997) J. Biol. Chem. 272, 13883–13891 16 Nare, B. et al. (1997) Parasitology 114, S101–S110 17 Hardy, L.W. et al. (1997) Exp. Parasitol. 87, 157–169 18 Eisenthal, R. and Cornish-Bowden, A. (1998) J. Biol. Chem. 273, 5500–5505 19 Perié, J. et al. (1993) Pharmacol. Ther. 60, 347–365 20 Dumas, C. et al. (1997) EMBO J. 16, 2590–2598 21 Tovar, J. et al. (1998) Mol. Microbiol. 29, 653–660 22 Tovar, J. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5311–5316 23 Wirtz, E. and Clayton, C.E. (1995) Science 268, 1179–1183 24 McKerrow, J.H. et al. (1993) Annu. Rev. Microbiol. 47, 821–853 25 Souza, A.E. et al. (1994) Mol. Biochem. Parasitol. 63, 213–220
26 Bart, G. et al. (1997) Mol. Biochem. Parasitol. 88, 53–61 27 Mottram, J.C. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6008–6013 28 Selzer, P.M. et al. (1997) Exp. Parasitol. 87, 212–221 29 Harth, G. et al. (1993) Mol. Biochem. Parasitol. 58, 17–24 30 Engel, J.C. et al. (1998) J. Cell Sci. 111, 597–606 31 Ullman, B. and Carter, D. (1995) Infect. Agents Dis. 4, 29–40 32 Hwang, H.Y. and Ullman, B. (1997) J. Biol. Chem. 272, 19488–19496 33 Hwang, H.Y. et al. (1996) J. Biol. Chem. 271, 30840–30846 34 Berens, R.L., Krug, E.C. and Marr, J.J. (1995) in Biochemistry and Molecular Biology of Parasites (Marr, J.J. and Muller, M., eds), pp. 89–117, Academic Press 35 Cully, D.F. et al. (1994) Nature 371, 707–711 36 Mottram, J.C., Brooks, D.R. and Coombs, G.H. (1998) Curr. Opin. Microbiol. 1, 455–460 37 Engel, J.C. et al. (1998) J. Exp. Med. 188, 725–734 38 Mbawa, Z.R. et al. (1992) Eur. J. Biochem. 204, 371–379 39 Joshi, P.B. et al. (1998) Mol. Microbiol. 27, 519–530 40 Webb, H. et al. (1997) J. Cell Biol. 139, 103–114
Proteasomes and other selfcompartmentalizing proteases in prokaryotes René De Mot, István Nagy, Jochen Walz and Wolfgang Baumeister
I
ntracellular proteases in pro- The proteasome represents the major non- ments in their interior (Fig. 1). karyotic cells perform many lysosomal proteolytic system in Narrow entrances to the cylintasks, including cleavage of eukaryotes. It confines proteolytic activity der restrict access to unfolded signal peptides during protein to an inner compartment that is accessible proteins. The ATPase comexport, timely inactivation of to unfolded proteins only. The strategy of plexes that can bind to both regulatory proteins, and removal controlling intracellular breakdown of ends of such barrels are thought of aberrant nonfunctional proproteins by self-compartmentalization is to be involved in the initial teins1. Obviously, proteolysis also used by different types of prokaryotic binding, unfolding and transenergy-dependent proteases. Genomic inside prokaryotic cells needs location of substrates. sequencing data reveal that various to be controlled to avoid uncombinations of these energy-dependent wanted degradation. Spatial Proteases creating proteases occur in prokaryotic cells from safeguarding can be provided nanocompartments by directing a protease to a spedifferent lineages. The proteasome, which has cific compartment of the cell, become the paradigm of a selfR. De Mot* and I. Nagy are in the F.A. Janssens such as the periplasmic space compartmentalizing protease3, voor Genetica, Katholieke Universiteit of Gram-negative bacteria. Laboratorium was first discovered in eukaryoLeuven, B-3001 Heverlee, Belgium; J. Walz and Autocompartmentalization, as tic cells, where it constitutes the W. Baumeister are in the Abteilung Molekulare a common strategy to curtail Strukturbiologie, Max-Planck-Institut für Biochemie, major non-lysosomal proteoD-82152 Martinsried, Germany. the potential hazard associated lytic system4. The proteolytic *tel: 132 16 329 681, with intracellular protein breakcore (20S proteasome), capped fax: 132 16 321 966, down, has recently emerged with 19S regulatory complexes e-mail: [email protected] from the elucidation of the (including several ATPases), is structure of a subset of cytodesignated the 26S proteasome plasmic proteases in prokaryotes2. Isolation of proteo- and mediates the ATP-dependent degradation of lytic activity is achieved by self-assembly of proteo- ubiquitin-bound proteins5. The archaeal counterpart lytic subunits into a cylinder-shaped complex, in of the 20S proteasome was identified in Thermoplasma which the active sites are confined to nanocompart- acidophilum about a decade ago6. The discovery of 0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
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this compositionally simpler complex (Fig. 1) has advanced our understanding of the novel mechanism involved in proteolysis, which is characteristic of amino-terminal nucleophile (Ntn) hydrolases7. This type of reaction involves autocatalytic removal of a propeptide, which exposes the amino-terminal active site residue, threonine. The first bacterial 20S proteasome was found in the actinomycete Rhodococcus erythropolis8,9. The genes of the prokaryote 20S proteasomes can be expressed in Escherichia coli without deleterious effects, and the gene products readily assemble into proteolytically active complexes with a native architecture. Other bacterial proteases that assemble into welldefined compartments have recently been characterized, namely ClpP and HslV from E. coli (Fig. 1). Unlike the 20S proteasome, which is composed of four heptameric rings, the proteolytic core of these proteases consists of only two rings that are either hexameric (HslV)10 or heptameric (ClpP)11. HslV belongs to the Ntn hydrolase family10,12, but ClpP is a serine protease. Degradation by HslV and ClpP is energy dependent and requires association with specific ATPases of the Clp family: HslU with HslV, and ClpX or ClpA with ClpP. These ATPases display intrinsic chaperone activity when not docked to their cognate protease component13. The ATPases that are part of the eukaryotic 19S complex belong to a different family, the AAA (ATPases associated with diverse cellular activities) family14. The characteristic AAA motif is also present in the membrane-bound E. coli metalloprotease FtsH (Ref. 1). The Lon protease represents another E. coli protease with intrinsic ATPase activity1. However, the structures of the FtsH and Lon proteases have not yet been defined. Distribution of 20S proteasomes in prokaryotes A comparative analysis of the complete genomic sequences of representative species from Archaea and Bacteria (overview at http://www.tigr.org/tigr_ home/tdb/mdb/mdb.html) provides information about the distribution of homologs of these proteases throughout different lineages (Table 1). Structural genes for 20S proteasomes are present in all archaeal genomes sequenced to date. Together with the isolation of proteasomes from T. acidophilum (Thermoplasmales)6, Methanosarcina thermophila (Methanomicrobiales)15 and Pyrococcus furiosus16, this highlights the widespread distribution of this protease, at least in Euryarchaeota. The availability of the genomic sequence of Sulfolobus solfataricus (Sulfolobales) should soon answer the question of whether 20S proteasomes also occur in the Crenarchaeota. However, the picture changes dramatically when surveying the Bacteria (Table 1). Genuine 20S proteasomes built of a- and b-type subunits apparently have a narrow range of distribution among bacteria. The presence of 20S proteasomes in Mycobacterium tuberculosis17, Mycobacterium leprae and Mycobacterium smegmatis18 was expected because both Mycobacterium and Rhodococcus belong to the group of mycolic-acid-containing actinomycetes. Recently, it
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Fig. 1. Surface models of self-compartmentalizing proteases obtained by low-pass filtering (1.2 nm cut-off) of the atomic models of (a) HslV (Ref. 10) and (b) ClpP (Ref. 11) from Escherichia coli, and (c) the 20S proteasome from Thermoplasma acidophilum32. For each structure, a side view (left), a cut-open side view with active sites marked by red spheres (center) and a top view (right) are shown. The position of the seven-membered rings composed of either a or b subunits are indicated for the 20S proteasome. The archaeal 20S proteasome contains 14 copies each of the a and b subunits (a7b7b7a7). A highly similar architecture is obtained for the yeast 20S proteasome by assembly of two copies each of 14 different subunits (seven of the a type and seven of the b type)33. Scale bar 5 10 nm.
has become clear that phylogenetically more distant actinomycetes, such as Streptomyces coelicolor, also contain this type of self-compartmentalizing protease19. Apart from Gram-positive bacteria with high GC content, no other bacteria are currently known to contain 20S proteasomes. Several species possess the proteasome-like system, HslVU; notable exceptions are the cyanobacterium Synechocystis sp. PCC6803, Mycoplasma genitalium, Mycoplasma pneumoniae, Chlamydia trachomatis and the spirochete Treponema pallidum. Homologs of hslV and hslU genes are present in another spirochete, Borrelia burgdorferi. The occurrence of 20S proteasome and HslVU systems is apparently mutually exclusive (Table 1). The currently recognized energy-dependent self-compartmentalizing proteases are absent from mycoplasmas, a group of cell-wall-less, low GC content Grampositive bacteria that have very small genomes. A striking feature of the M. tuberculosis H37Rv genome is the absence of a gene for the ubiquitous Lon protease17, particularly because a Lon protease has recently been identified in the non-pathogenic, fast-growing M. smegmatis20. It will be of interest to see from the other mycobacterial genomes currently
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Table 1. Energy-dependent proteases in microbial genomesa Domain
Order/lineage
Species
20S proteasome
HslVU
ClpAP/ ClpXP
Lon
FtsH
Archaea
Archaeoglobales Methanococcales Methanobacteriales
Archaeoglobus fulgidus Methanococcus jannaschii Methanobacterium thermoautotrophicum Pyrococcus horikoshii Mycobacterium tuberculosis Aquifex aeolicus Chlamydia trachomatis Synechocystis sp. PCC6803 Bacillus subtilis Mycoplasma genitalium Mycoplasma pneumoniae Escherichia coli Haemophilus influenzae Helicobacter pylori Rickettsia prowazekii Borrelia burgdorferi Treponema pallidum Saccharomyces cerevisiae
1 1 1
2 2 2
2 2 2
1 1 1
2 2 2
1(2) 1b 2 2 2 2 2 2 2 2 2 2 2 2 1(3)d
2 2 1 2 2 1 2 2 1 1 1 1 1 2 2
2 1(2) 1 1(2) 1(4) 1 2 2 1 1 1 1 1(2) 1(2) 2e
1 2c 1 1 2 1(2) 1 1 1 1 1 1 1(2) 1(2) 1
2 1 1 1 1(4) 1 1 1 1 1 1 1 1 1 1
Bacteria
Thermococcales Actinomycetes Aquificales Chlamydiales Cyanobacteria Low-GC Gram-positive Mycoplasmas Proteobacteria
Spirochetes Eukarya
Fungi
a
The presence (1) or absence (2) of genes for energy-dependent proteases in completely sequenced microbial genomes is indicated. The numbers in parentheses refer to the occurrence of multiple genes for a particular type of protease (Lon or FtsH) or proteolytic subunit in self-compartmentalizing proteases (proteasome b subunit, HslV or ClpP). b Two different b-type subunits are present in the proteasomes of the related actinomycete Rhodococcus erythropolis NI86/21 (Refs 8,9), but a single b-type subunit gene is found in several other R. erythropolis strains and other Rhodococcus species (I. Nagy et al., unpublished). c A functional Lon homolog exists in Mycobacterium smegmatis20. d Eukaryal 20S proteasomes contain seven different b-type subunits, only three of which are proteolytically active3. e ClpP homologs are present in chloroplasts and in human mitochondria.
being sequenced whether the absence/presence of a Lon homolog somehow correlates with growth rate and/or pathogenicity in this genus. A gene encoding a Lon homolog is also absent from the Synechocystis sp. PCC6803 genome. Energy dependence of 20S proteasomes Interaction of prokaryotic 20S proteasomes with ATPases is anticipated but has not yet been demonstrated. Relatives of the AAA-type ATPases present in the 19S caps of the eukaryote proteasome (26S proteasome branch of the AAA tree14; http://yeamob. pci.chemie.uni-tuebingen.de/AAA/) are emerging from all sequenced archaeal genomes and are primary candidates for possible interaction with 20S proteasomes. A divergent AAA ATPase is encoded in the upstream region of proteasome genes in M. leprae, M. tuberculosis17, R. erythropolis21 and S. coelicolor19. A predicted coiled-coil segment near the amino terminus is a hallmark of 19S cap ATPases and their archaeal relatives and is also a characteristic of the actinomycete ATPases. The Rhodococcus protein, ARC (AAA ATPase forming ring-shaped complexes), assembles into six-membered ring structures with ATPase activity, but its predicted interaction with bacterial 20S proteasomes has yet to be demonstrated21. In addition to FtsH (metalloprotease branch14) and the ARC homolog Rv2115c (novel branch), another member of the AAA family, Rv0435c, is encoded by
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the M. tuberculosis genome17. This protein shows significant homology to several archaeal proteins, which contain two AAA ATPase domains and belong to the cell division/membrane fusion branch within the AAA family14. Interestingly, neither ARC nor Rv0435c homologs have been identified in bacteria lacking 20S proteasomes. Similarities and differences between 20S proteasomes In eukaryote 20S proteasomes, three out of seven different b-type subunits are active and confer three major types of proteolytic activity3. Only chymotrypticlike activity is detectable with prokaryotic 20S proteasomes and HslVU. In prokaryotic 20S proteasomes, all b-type subunits are active3. This also applies to the 20S proteasome from R. erythropolis NI86/21 (Ref. 22), the only bacterial proteasome known to be built from two different b-type subunits (and two different a-type subunits)8. Zühl et al.9 have shown that different combinations of these a- and b-type subunits result in 20S proteasomes with slightly changed kinetic parameters. The most recent addition to the list of sequenced archaeal genomes suggests that Pyrococcus horikoshii proteasomes might also contain two different b-type subunits, but only one type of a subunit. The (unfinished) sequencing of the P. furiosus genome confirms the existence of two distinct genes for b-type subunits in this genus
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(http://www.ncbi.nlm.nih.gov/BLAST/ unfinishedgenome.html). The co-expression of both b subunit genes needs to be confirmed, as only one of the b-type subunits has been identified in purified 20S proteasomes from P. furiosus DSM3638 (Ref. 16). Such redundancy of genes for proteases or proteolytic subunits is apparent for other energy-dependent proteases in several bacterial genomes (Table 1). This is most prominent in Synechocystis sp. PCC6803, which has four homologs each of ClpP and FtsH. M. tuberculosis H37Rv contains two linked clpP genes that seem to be translationally coupled (overlap of respective stop and initiation codons)17. Translational coupling of the 20S proteasome genes in M. leprae, M. smegmatis, M. tuberculosis and R. erythropolis is believed to be involved in equimolar expression of b and a subunits8. Several differences have been observed in the assembly pathways of archaeal and bacterial 20S proteasomes3. In T. acidophilum, an a ring serves as a template for b subunit assembly23, whereas a/b dimers are probably early intermediates in R. erythropolis24. Assembly proceeds via inactive half-proteasomes that then dimerize and are activated by autocatalytic cleavage of b propeptides. The archaeal b propeptides (,10 amino acids) and putative propeptides of some HslV homologs (,7 amino acids) are considerably shorter than those of bacteria (up to 65 amino acids). The Rhodococcus propeptides act as intramolecular chaperones, promoting folding of b subunits and further maturation of half-proteasomes24. Sequence homology between b propeptides from bacterial species is low and confined to a short central region and a conserved penultimate His residue preceding the invariant Gly–Thr cleavage site19. The size of bacterial b propeptides is reminiscent of some of their eukaryotic counterparts, which also display chaperone-like activity3. Functions for prokaryotic proteasomes In Eukarya, proteasomes are involved in many different important cellular processes, ranging from cellcycle control to antigen processing. Cellular targets for 20S proteasomes in prokaryotes remain to be identified. For a proteasome knockout mutant of M. smegmatis, no discernable phenotypic changes, including responses to stress conditions, have been identified18. However, specific inactivation of most of the 20S proteasome activity of T. acidophilum by a vinyl sulfone inhibitor severely impairs cell viability following heat-shock treatment25. A possible explanation might be that other proteases compensate for the loss of proteasomes in bacteria, but that such a back-up system to cope with stress conditions is absent or less efficient in Archaea. In E. coli, HslVU is induced by heat shock and acts synergistically with the other ATP-dependent proteases to control turnover of the sigma factor RpoH (s32) in vivo. The transiently increased level of RpoH directs transcription of heat-shock genes26. No such heat-shock dependence of proteasome production is obvious in actinomycetes (I. Nagy et al., unpublished). As a Lon protease is apparently absent from M. tuberculosis, disabling of the
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Questions for future research • What are the cellular targets for the various self-compartmentalizing proteases in prokaryotic cells, and are specific tagging systems involved? When different self-compartmentalizing proteases occur in a single cell, are these systems redundant or do they act independently on specific subsets of substrates? • What types of ATP-dependent systems operate in conjunction with the prokaryotic 20S proteasomes? • Are the genes that are part of a conserved operon-like structure, together with the 20S proteasome structural genes in bacteria, involved in proteasome functioning? • Do proteasome-inhibitory metabolites from actinomycetes (such as lactacystin) affect proteasome activity in the producer cells? If not, how do the cells prevent their proteasomes from getting irreversibly inactivated? How widespread is the production of proteasome inhibitors among actinomycetes? • Could secretion of a potent proteasome inhibitor provide a competitive advantage to bacteria in natural niches such as soil? Is there a role for bacterial proteasome inhibitors, during infection of hosts by pathogens (such as Mycobacterium) or when establishing symbioses with plants (for example, by nitrogenfixing Frankia), by modulating proteasome activity in eukaryotic cells?
proteasome might engender more dramatic effects on cellular physiology (and possibly virulence) than would be anticipated from the phenotypic characterization of the corresponding M. smegmatis mutant18. In addition to gene knockouts, the use of specific inhibitors will probably become important in elucidating the cellular functions of prokaryotic proteasomes. One such inhibitor is lactacystin27, which inactivates the 20S proteasomes of both Rhodococcus22 and Streptomyces19 by covalent modification of the active site threonines. Remarkably, lactacystin is a metabolite secreted by Streptomyces sp. OM-6519. It will be of interest to investigate how the production of a potent proteasome inhibitor is reconciled with the presence of proteasomes. It is not known whether the proteasome system in prokaryotes requires some kind of protein-tagging system equivalent to the eukaryotic ubiquitin system. Ubiquitin genes are absent from all prokaryotic genomes sequenced so far, but other tagging systems might be involved. The modification of amino-terminal residues, mediated by Aat (a Leu/Phe-tRNA-protein transferase), and subsequent degradation by ClpAP represents a system of amino-terminal recognition in E. coli. Functional Aat homologs have been identified in some other bacteria, including Synechocystis sp.28 Carboxy-terminal peptide tags incorporated via the 10Sa RNA (tmRNA) in E. coli are recognized by the periplasmic protease Tsp (Ref. 29), the cytoplasmic proteases ClpXP and ClpAP (Ref. 30) and the membrane-bound FtsH (Ref. 31), but recognition by the proteasome-like HslVU has not yet been demonstrated. Homologs of the tmRNA gene ssrA have been identified in many bacteria (http://www.wi.mit. edu/bartel/tmRNA/home) and Tsp-like proteases are potentially encoded by most bacterial genomes, which indicates that such tagging mechanisms might be common among bacteria.
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Acknowledgements This work is supported by a grant from the Fund for Scientific Research (Flanders) to R.D.M. We thank P. Zwickl for critical reading of the manuscript and M-N. Pouch for communicating unpublished data. References 1 Gottesman, S. (1996) Annu. Rev. Genet. 30, 465–506 2 Lupas, A. et al. (1997) Trends Biochem. Sci. 22, 399–404 3 Baumeister, W. et al. (1998) Cell 92, 367–380 4 Coux, O., Tanaka, K. and Goldberg, A.L. (1996) Annu. Rev. Biochem. 65, 801–847 5 Varshavsky, A. (1997) Trends Biochem. Sci. 22, 383–387 6 Dahlmann, B. et al. (1989) FEBS Lett. 251, 125–131 7 Seemüller, E. et al. (1995) Science 268, 579–582 8 Tamura, T. et al. (1995) Curr. Biol. 5, 766–774 9 Zühl, F. et al. (1997) FEBS Lett. 400, 83–90 10 Bochtler, M. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6070–6074 11 Wang, J., Hartling, J.A. and Flanagan, J.M. (1997) Cell 91, 447–456 12 Bogyo, M. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6629–6634 13 Gottesman, S., Maurizi, M.R. and Wickner, S. (1997) Cell 91, 435–438 14 Patel, S. and Latterich, M. (1998) Trends Cell Biol. 8, 65–71 15 Maupin-Furlow, J.A. and Ferry, J.G. (1995) J. Biol. Chem. 270, 28617–28622
Magic mushrooms Fungal Morphogenesis by D. Moore Cambridge University Press, 1998. £70.00 hbk (xiv 1 469 pages) ISBN 0 521 55295 8
F
ungal Morphogenesis is a rare example of a single author book that covers a large and developing area. The author is an established authority on the developmental biology of mushrooms of higher fungi. This area is certainly the comfort zone of the book, and slightly over half of the text is devoted to it, despite the fact that it is a somewhat neglected area by developmental biologists. A lot of what is known about fruit body formation is descriptive, although a few well-characterized aspects of the biology of these organisms have been developed recently. For example, the analysis of mating type genes, gene expression in homokaryotic and dikaryotic mycelia and the role of hydrophobins in the development of aerial structures have all advanced considerably in recent years and are well covered here. The author also shows how growth of the
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16 Bauer, M.W., Bauer, S.H. and Kelly, R.M. (1997) Appl. Environ. Microbiol. 63, 1160–1164 17 Cole, S.T. et al. (1998) Nature 393, 537–544 18 Knipfer, N. and Shrader, T.E. (1997) Mol. Microbiol. 25, 375–383 19 Nagy, I. et al. (1998) J. Bacteriol. 180, 5448–5453 20 Roudiak, S.G. et al. (1998) Biochemistry 37, 377–386 21 Wolf, S. et al. (1998) J. Mol. Biol. 277, 13–25 22 McCormack, T. et al. (1997) J. Biol. Chem. 272, 26103–26109 23 Seemüller, E., Lupas, A. and Baumeister, W. (1996) Nature 382, 468–470 24 Zühl, F. et al. (1997) FEBS Lett. 418, 189–194 25 Ruepp, A. et al. (1998) FEBS Lett. 425, 87–90 26 Kanemori, M. et al. (1997) J. Bacteriol. 179, 7219–7225 27 Fenteany, G. and Schreiber, S.L. (1998) J. Biol. Chem. 273, 8545–8548 28 Ichetovkin, I.E., Abramochkin, G. and Shrader, T.E. (1997) J. Biol. Chem. 272, 33009–33014 29 Keiler, K.C., Patrick, R.H. and Sauer, R.T. (1996) Science 271, 990–993 30 Gottesman, S. et al. (1998) Genes Dev. 12, 1338–1347 31 Herman, C. et al. (1998) Genes Dev. 12, 1348–1355 32 Löwe, J. et al. (1995) Science 268, 533–539 33 Groll, M. et al. (1997) Nature 386, 463–471
basidiocarp is regulated so that the spore-bearing gills are presented in optimal alignment for efficient liberation of spores into the environment. Material is brought together from a range of sources and approaches to provide a useful overview of the present status of the field as a whole and to highlight hot topics for the future. As for the rest of the book, I must confess to being a little disappointed about what is left out. The recent strides forward in our understanding of the regulation and molecular events concerned with cell polarity and bud site selection and the interplay between cell cycle regulation and bud morphogenesis (all in yeast) are not mentioned. The regulation of pseudohyphal and hyphal growth in yeasts is covered in only a paragraph, and recent molecular genetic analyses of hyphal growth, septum formation and branching in Aspergillus are barely touched on. These studies have pushed fungal morphogenesis to the forefront of developmental biology and have been reported in the most prestigious journals. To my mind, they are of profound importance to our appreciation of, for example, hyphal tip growth, branch for-
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mation and clamp connections in all fungi and, as such, I would have expected them to have been a major part of the book. Perhaps the omission of these key areas is the result of the strong focus on basidiomycete development. The book also has a whole chapter devoted to metabolism and biochemistry, with figures showing all the major metabolic pathways. Fine, but these pathways are generally common to most life forms and cannot be seen as essential to the theme of the book. Perhaps it was a tall order to get the broad coverage that I anticipated from the title. I can see this book being highly recommended for those working on, or wanting to know more about, the morphogenesis of higher fungi. However, those of a molecular genetic bent who are interested in a wider diversity of model fungal systems or the general molecular mechanisms underlying morphogenesis will feel less well served. Neil Gow Dept of Molecular and Cell Biology, Marischal College, University of Aberdeen, Aberdeen, UK AB25 2ZD.
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12 Cruz, A.K., Coburn, C.M. and Beverley, S.M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7170–7174 13 Titus, R.G. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10267–10271 14 Gueiros-Filho, F.J. and Beverley, S.M. (1996) Mol. Cell. Biol. 16, 5655–5663 15 Nare, B., Hardy, L.W. and Beverley, S.M. (1997) J. Biol. Chem. 272, 13883–13891 16 Nare, B. et al. (1997) Parasitology 114, S101–S110 17 Hardy, L.W. et al. (1997) Exp. Parasitol. 87, 157–169 18 Eisenthal, R. and Cornish-Bowden, A. (1998) J. Biol. Chem. 273, 5500–5505 19 Perié, J. et al. (1993) Pharmacol. Ther. 60, 347–365 20 Dumas, C. et al. (1997) EMBO J. 16, 2590–2598 21 Tovar, J. et al. (1998) Mol. Microbiol. 29, 653–660 22 Tovar, J. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5311–5316 23 Wirtz, E. and Clayton, C.E. (1995) Science 268, 1179–1183 24 McKerrow, J.H. et al. (1993) Annu. Rev. Microbiol. 47, 821–853 25 Souza, A.E. et al. (1994) Mol. Biochem. Parasitol. 63, 213–220
26 Bart, G. et al. (1997) Mol. Biochem. Parasitol. 88, 53–61 27 Mottram, J.C. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6008–6013 28 Selzer, P.M. et al. (1997) Exp. Parasitol. 87, 212–221 29 Harth, G. et al. (1993) Mol. Biochem. Parasitol. 58, 17–24 30 Engel, J.C. et al. (1998) J. Cell Sci. 111, 597–606 31 Ullman, B. and Carter, D. (1995) Infect. Agents Dis. 4, 29–40 32 Hwang, H.Y. and Ullman, B. (1997) J. Biol. Chem. 272, 19488–19496 33 Hwang, H.Y. et al. (1996) J. Biol. Chem. 271, 30840–30846 34 Berens, R.L., Krug, E.C. and Marr, J.J. (1995) in Biochemistry and Molecular Biology of Parasites (Marr, J.J. and Muller, M., eds), pp. 89–117, Academic Press 35 Cully, D.F. et al. (1994) Nature 371, 707–711 36 Mottram, J.C., Brooks, D.R. and Coombs, G.H. (1998) Curr. Opin. Microbiol. 1, 455–460 37 Engel, J.C. et al. (1998) J. Exp. Med. 188, 725–734 38 Mbawa, Z.R. et al. (1992) Eur. J. Biochem. 204, 371–379 39 Joshi, P.B. et al. (1998) Mol. Microbiol. 27, 519–530 40 Webb, H. et al. (1997) J. Cell Biol. 139, 103–114
Proteasomes and other selfcompartmentalizing proteases in prokaryotes René De Mot, István Nagy, Jochen Walz and Wolfgang Baumeister
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ntracellular proteases in pro- The proteasome represents the major non- ments in their interior (Fig. 1). karyotic cells perform many lysosomal proteolytic system in Narrow entrances to the cylintasks, including cleavage of eukaryotes. It confines proteolytic activity der restrict access to unfolded signal peptides during protein to an inner compartment that is accessible proteins. The ATPase comexport, timely inactivation of to unfolded proteins only. The strategy of plexes that can bind to both regulatory proteins, and removal controlling intracellular breakdown of ends of such barrels are thought of aberrant nonfunctional proproteins by self-compartmentalization is to be involved in the initial teins1. Obviously, proteolysis also used by different types of prokaryotic binding, unfolding and transenergy-dependent proteases. Genomic inside prokaryotic cells needs location of substrates. sequencing data reveal that various to be controlled to avoid uncombinations of these energy-dependent wanted degradation. Spatial Proteases creating proteases occur in prokaryotic cells from safeguarding can be provided nanocompartments by directing a protease to a spedifferent lineages. The proteasome, which has cific compartment of the cell, become the paradigm of a selfR. De Mot* and I. Nagy are in the F.A. Janssens such as the periplasmic space compartmentalizing protease3, voor Genetica, Katholieke Universiteit of Gram-negative bacteria. Laboratorium was first discovered in eukaryoLeuven, B-3001 Heverlee, Belgium; J. Walz and Autocompartmentalization, as tic cells, where it constitutes the W. Baumeister are in the Abteilung Molekulare a common strategy to curtail Strukturbiologie, Max-Planck-Institut für Biochemie, major non-lysosomal proteoD-82152 Martinsried, Germany. the potential hazard associated lytic system4. The proteolytic *tel: 132 16 329 681, with intracellular protein breakcore (20S proteasome), capped fax: 132 16 321 966, down, has recently emerged with 19S regulatory complexes e-mail: [email protected] from the elucidation of the (including several ATPases), is structure of a subset of cytodesignated the 26S proteasome plasmic proteases in prokaryotes2. Isolation of proteo- and mediates the ATP-dependent degradation of lytic activity is achieved by self-assembly of proteo- ubiquitin-bound proteins5. The archaeal counterpart lytic subunits into a cylinder-shaped complex, in of the 20S proteasome was identified in Thermoplasma which the active sites are confined to nanocompart- acidophilum about a decade ago6. The discovery of 0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
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this compositionally simpler complex (Fig. 1) has advanced our understanding of the novel mechanism involved in proteolysis, which is characteristic of amino-terminal nucleophile (Ntn) hydrolases7. This type of reaction involves autocatalytic removal of a propeptide, which exposes the amino-terminal active site residue, threonine. The first bacterial 20S proteasome was found in the actinomycete Rhodococcus erythropolis8,9. The genes of the prokaryote 20S proteasomes can be expressed in Escherichia coli without deleterious effects, and the gene products readily assemble into proteolytically active complexes with a native architecture. Other bacterial proteases that assemble into welldefined compartments have recently been characterized, namely ClpP and HslV from E. coli (Fig. 1). Unlike the 20S proteasome, which is composed of four heptameric rings, the proteolytic core of these proteases consists of only two rings that are either hexameric (HslV)10 or heptameric (ClpP)11. HslV belongs to the Ntn hydrolase family10,12, but ClpP is a serine protease. Degradation by HslV and ClpP is energy dependent and requires association with specific ATPases of the Clp family: HslU with HslV, and ClpX or ClpA with ClpP. These ATPases display intrinsic chaperone activity when not docked to their cognate protease component13. The ATPases that are part of the eukaryotic 19S complex belong to a different family, the AAA (ATPases associated with diverse cellular activities) family14. The characteristic AAA motif is also present in the membrane-bound E. coli metalloprotease FtsH (Ref. 1). The Lon protease represents another E. coli protease with intrinsic ATPase activity1. However, the structures of the FtsH and Lon proteases have not yet been defined. Distribution of 20S proteasomes in prokaryotes A comparative analysis of the complete genomic sequences of representative species from Archaea and Bacteria (overview at http://www.tigr.org/tigr_ home/tdb/mdb/mdb.html) provides information about the distribution of homologs of these proteases throughout different lineages (Table 1). Structural genes for 20S proteasomes are present in all archaeal genomes sequenced to date. Together with the isolation of proteasomes from T. acidophilum (Thermoplasmales)6, Methanosarcina thermophila (Methanomicrobiales)15 and Pyrococcus furiosus16, this highlights the widespread distribution of this protease, at least in Euryarchaeota. The availability of the genomic sequence of Sulfolobus solfataricus (Sulfolobales) should soon answer the question of whether 20S proteasomes also occur in the Crenarchaeota. However, the picture changes dramatically when surveying the Bacteria (Table 1). Genuine 20S proteasomes built of a- and b-type subunits apparently have a narrow range of distribution among bacteria. The presence of 20S proteasomes in Mycobacterium tuberculosis17, Mycobacterium leprae and Mycobacterium smegmatis18 was expected because both Mycobacterium and Rhodococcus belong to the group of mycolic-acid-containing actinomycetes. Recently, it
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Trends in Microbiology
Fig. 1. Surface models of self-compartmentalizing proteases obtained by low-pass filtering (1.2 nm cut-off) of the atomic models of (a) HslV (Ref. 10) and (b) ClpP (Ref. 11) from Escherichia coli, and (c) the 20S proteasome from Thermoplasma acidophilum32. For each structure, a side view (left), a cut-open side view with active sites marked by red spheres (center) and a top view (right) are shown. The position of the seven-membered rings composed of either a or b subunits are indicated for the 20S proteasome. The archaeal 20S proteasome contains 14 copies each of the a and b subunits (a7b7b7a7). A highly similar architecture is obtained for the yeast 20S proteasome by assembly of two copies each of 14 different subunits (seven of the a type and seven of the b type)33. Scale bar 5 10 nm.
has become clear that phylogenetically more distant actinomycetes, such as Streptomyces coelicolor, also contain this type of self-compartmentalizing protease19. Apart from Gram-positive bacteria with high GC content, no other bacteria are currently known to contain 20S proteasomes. Several species possess the proteasome-like system, HslVU; notable exceptions are the cyanobacterium Synechocystis sp. PCC6803, Mycoplasma genitalium, Mycoplasma pneumoniae, Chlamydia trachomatis and the spirochete Treponema pallidum. Homologs of hslV and hslU genes are present in another spirochete, Borrelia burgdorferi. The occurrence of 20S proteasome and HslVU systems is apparently mutually exclusive (Table 1). The currently recognized energy-dependent self-compartmentalizing proteases are absent from mycoplasmas, a group of cell-wall-less, low GC content Grampositive bacteria that have very small genomes. A striking feature of the M. tuberculosis H37Rv genome is the absence of a gene for the ubiquitous Lon protease17, particularly because a Lon protease has recently been identified in the non-pathogenic, fast-growing M. smegmatis20. It will be of interest to see from the other mycobacterial genomes currently
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Table 1. Energy-dependent proteases in microbial genomesa Domain
Order/lineage
Species
20S proteasome
HslVU
ClpAP/ ClpXP
Lon
FtsH
Archaea
Archaeoglobales Methanococcales Methanobacteriales
Archaeoglobus fulgidus Methanococcus jannaschii Methanobacterium thermoautotrophicum Pyrococcus horikoshii Mycobacterium tuberculosis Aquifex aeolicus Chlamydia trachomatis Synechocystis sp. PCC6803 Bacillus subtilis Mycoplasma genitalium Mycoplasma pneumoniae Escherichia coli Haemophilus influenzae Helicobacter pylori Rickettsia prowazekii Borrelia burgdorferi Treponema pallidum Saccharomyces cerevisiae
1 1 1
2 2 2
2 2 2
1 1 1
2 2 2
1(2) 1b 2 2 2 2 2 2 2 2 2 2 2 2 1(3)d
2 2 1 2 2 1 2 2 1 1 1 1 1 2 2
2 1(2) 1 1(2) 1(4) 1 2 2 1 1 1 1 1(2) 1(2) 2e
1 2c 1 1 2 1(2) 1 1 1 1 1 1 1(2) 1(2) 1
2 1 1 1 1(4) 1 1 1 1 1 1 1 1 1 1
Bacteria
Thermococcales Actinomycetes Aquificales Chlamydiales Cyanobacteria Low-GC Gram-positive Mycoplasmas Proteobacteria
Spirochetes Eukarya
Fungi
a
The presence (1) or absence (2) of genes for energy-dependent proteases in completely sequenced microbial genomes is indicated. The numbers in parentheses refer to the occurrence of multiple genes for a particular type of protease (Lon or FtsH) or proteolytic subunit in self-compartmentalizing proteases (proteasome b subunit, HslV or ClpP). b Two different b-type subunits are present in the proteasomes of the related actinomycete Rhodococcus erythropolis NI86/21 (Refs 8,9), but a single b-type subunit gene is found in several other R. erythropolis strains and other Rhodococcus species (I. Nagy et al., unpublished). c A functional Lon homolog exists in Mycobacterium smegmatis20. d Eukaryal 20S proteasomes contain seven different b-type subunits, only three of which are proteolytically active3. e ClpP homologs are present in chloroplasts and in human mitochondria.
being sequenced whether the absence/presence of a Lon homolog somehow correlates with growth rate and/or pathogenicity in this genus. A gene encoding a Lon homolog is also absent from the Synechocystis sp. PCC6803 genome. Energy dependence of 20S proteasomes Interaction of prokaryotic 20S proteasomes with ATPases is anticipated but has not yet been demonstrated. Relatives of the AAA-type ATPases present in the 19S caps of the eukaryote proteasome (26S proteasome branch of the AAA tree14; http://yeamob. pci.chemie.uni-tuebingen.de/AAA/) are emerging from all sequenced archaeal genomes and are primary candidates for possible interaction with 20S proteasomes. A divergent AAA ATPase is encoded in the upstream region of proteasome genes in M. leprae, M. tuberculosis17, R. erythropolis21 and S. coelicolor19. A predicted coiled-coil segment near the amino terminus is a hallmark of 19S cap ATPases and their archaeal relatives and is also a characteristic of the actinomycete ATPases. The Rhodococcus protein, ARC (AAA ATPase forming ring-shaped complexes), assembles into six-membered ring structures with ATPase activity, but its predicted interaction with bacterial 20S proteasomes has yet to be demonstrated21. In addition to FtsH (metalloprotease branch14) and the ARC homolog Rv2115c (novel branch), another member of the AAA family, Rv0435c, is encoded by
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the M. tuberculosis genome17. This protein shows significant homology to several archaeal proteins, which contain two AAA ATPase domains and belong to the cell division/membrane fusion branch within the AAA family14. Interestingly, neither ARC nor Rv0435c homologs have been identified in bacteria lacking 20S proteasomes. Similarities and differences between 20S proteasomes In eukaryote 20S proteasomes, three out of seven different b-type subunits are active and confer three major types of proteolytic activity3. Only chymotrypticlike activity is detectable with prokaryotic 20S proteasomes and HslVU. In prokaryotic 20S proteasomes, all b-type subunits are active3. This also applies to the 20S proteasome from R. erythropolis NI86/21 (Ref. 22), the only bacterial proteasome known to be built from two different b-type subunits (and two different a-type subunits)8. Zühl et al.9 have shown that different combinations of these a- and b-type subunits result in 20S proteasomes with slightly changed kinetic parameters. The most recent addition to the list of sequenced archaeal genomes suggests that Pyrococcus horikoshii proteasomes might also contain two different b-type subunits, but only one type of a subunit. The (unfinished) sequencing of the P. furiosus genome confirms the existence of two distinct genes for b-type subunits in this genus
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(http://www.ncbi.nlm.nih.gov/BLAST/ unfinishedgenome.html). The co-expression of both b subunit genes needs to be confirmed, as only one of the b-type subunits has been identified in purified 20S proteasomes from P. furiosus DSM3638 (Ref. 16). Such redundancy of genes for proteases or proteolytic subunits is apparent for other energy-dependent proteases in several bacterial genomes (Table 1). This is most prominent in Synechocystis sp. PCC6803, which has four homologs each of ClpP and FtsH. M. tuberculosis H37Rv contains two linked clpP genes that seem to be translationally coupled (overlap of respective stop and initiation codons)17. Translational coupling of the 20S proteasome genes in M. leprae, M. smegmatis, M. tuberculosis and R. erythropolis is believed to be involved in equimolar expression of b and a subunits8. Several differences have been observed in the assembly pathways of archaeal and bacterial 20S proteasomes3. In T. acidophilum, an a ring serves as a template for b subunit assembly23, whereas a/b dimers are probably early intermediates in R. erythropolis24. Assembly proceeds via inactive half-proteasomes that then dimerize and are activated by autocatalytic cleavage of b propeptides. The archaeal b propeptides (,10 amino acids) and putative propeptides of some HslV homologs (,7 amino acids) are considerably shorter than those of bacteria (up to 65 amino acids). The Rhodococcus propeptides act as intramolecular chaperones, promoting folding of b subunits and further maturation of half-proteasomes24. Sequence homology between b propeptides from bacterial species is low and confined to a short central region and a conserved penultimate His residue preceding the invariant Gly–Thr cleavage site19. The size of bacterial b propeptides is reminiscent of some of their eukaryotic counterparts, which also display chaperone-like activity3. Functions for prokaryotic proteasomes In Eukarya, proteasomes are involved in many different important cellular processes, ranging from cellcycle control to antigen processing. Cellular targets for 20S proteasomes in prokaryotes remain to be identified. For a proteasome knockout mutant of M. smegmatis, no discernable phenotypic changes, including responses to stress conditions, have been identified18. However, specific inactivation of most of the 20S proteasome activity of T. acidophilum by a vinyl sulfone inhibitor severely impairs cell viability following heat-shock treatment25. A possible explanation might be that other proteases compensate for the loss of proteasomes in bacteria, but that such a back-up system to cope with stress conditions is absent or less efficient in Archaea. In E. coli, HslVU is induced by heat shock and acts synergistically with the other ATP-dependent proteases to control turnover of the sigma factor RpoH (s32) in vivo. The transiently increased level of RpoH directs transcription of heat-shock genes26. No such heat-shock dependence of proteasome production is obvious in actinomycetes (I. Nagy et al., unpublished). As a Lon protease is apparently absent from M. tuberculosis, disabling of the
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Questions for future research • What are the cellular targets for the various self-compartmentalizing proteases in prokaryotic cells, and are specific tagging systems involved? When different self-compartmentalizing proteases occur in a single cell, are these systems redundant or do they act independently on specific subsets of substrates? • What types of ATP-dependent systems operate in conjunction with the prokaryotic 20S proteasomes? • Are the genes that are part of a conserved operon-like structure, together with the 20S proteasome structural genes in bacteria, involved in proteasome functioning? • Do proteasome-inhibitory metabolites from actinomycetes (such as lactacystin) affect proteasome activity in the producer cells? If not, how do the cells prevent their proteasomes from getting irreversibly inactivated? How widespread is the production of proteasome inhibitors among actinomycetes? • Could secretion of a potent proteasome inhibitor provide a competitive advantage to bacteria in natural niches such as soil? Is there a role for bacterial proteasome inhibitors, during infection of hosts by pathogens (such as Mycobacterium) or when establishing symbioses with plants (for example, by nitrogenfixing Frankia), by modulating proteasome activity in eukaryotic cells?
proteasome might engender more dramatic effects on cellular physiology (and possibly virulence) than would be anticipated from the phenotypic characterization of the corresponding M. smegmatis mutant18. In addition to gene knockouts, the use of specific inhibitors will probably become important in elucidating the cellular functions of prokaryotic proteasomes. One such inhibitor is lactacystin27, which inactivates the 20S proteasomes of both Rhodococcus22 and Streptomyces19 by covalent modification of the active site threonines. Remarkably, lactacystin is a metabolite secreted by Streptomyces sp. OM-6519. It will be of interest to investigate how the production of a potent proteasome inhibitor is reconciled with the presence of proteasomes. It is not known whether the proteasome system in prokaryotes requires some kind of protein-tagging system equivalent to the eukaryotic ubiquitin system. Ubiquitin genes are absent from all prokaryotic genomes sequenced so far, but other tagging systems might be involved. The modification of amino-terminal residues, mediated by Aat (a Leu/Phe-tRNA-protein transferase), and subsequent degradation by ClpAP represents a system of amino-terminal recognition in E. coli. Functional Aat homologs have been identified in some other bacteria, including Synechocystis sp.28 Carboxy-terminal peptide tags incorporated via the 10Sa RNA (tmRNA) in E. coli are recognized by the periplasmic protease Tsp (Ref. 29), the cytoplasmic proteases ClpXP and ClpAP (Ref. 30) and the membrane-bound FtsH (Ref. 31), but recognition by the proteasome-like HslVU has not yet been demonstrated. Homologs of the tmRNA gene ssrA have been identified in many bacteria (http://www.wi.mit. edu/bartel/tmRNA/home) and Tsp-like proteases are potentially encoded by most bacterial genomes, which indicates that such tagging mechanisms might be common among bacteria.
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Acknowledgements This work is supported by a grant from the Fund for Scientific Research (Flanders) to R.D.M. We thank P. Zwickl for critical reading of the manuscript and M-N. Pouch for communicating unpublished data. References 1 Gottesman, S. (1996) Annu. Rev. Genet. 30, 465–506 2 Lupas, A. et al. (1997) Trends Biochem. Sci. 22, 399–404 3 Baumeister, W. et al. (1998) Cell 92, 367–380 4 Coux, O., Tanaka, K. and Goldberg, A.L. (1996) Annu. Rev. Biochem. 65, 801–847 5 Varshavsky, A. (1997) Trends Biochem. Sci. 22, 383–387 6 Dahlmann, B. et al. (1989) FEBS Lett. 251, 125–131 7 Seemüller, E. et al. (1995) Science 268, 579–582 8 Tamura, T. et al. (1995) Curr. Biol. 5, 766–774 9 Zühl, F. et al. (1997) FEBS Lett. 400, 83–90 10 Bochtler, M. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6070–6074 11 Wang, J., Hartling, J.A. and Flanagan, J.M. (1997) Cell 91, 447–456 12 Bogyo, M. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6629–6634 13 Gottesman, S., Maurizi, M.R. and Wickner, S. (1997) Cell 91, 435–438 14 Patel, S. and Latterich, M. (1998) Trends Cell Biol. 8, 65–71 15 Maupin-Furlow, J.A. and Ferry, J.G. (1995) J. Biol. Chem. 270, 28617–28622
Magic mushrooms Fungal Morphogenesis by D. Moore Cambridge University Press, 1998. £70.00 hbk (xiv 1 469 pages) ISBN 0 521 55295 8
F
ungal Morphogenesis is a rare example of a single author book that covers a large and developing area. The author is an established authority on the developmental biology of mushrooms of higher fungi. This area is certainly the comfort zone of the book, and slightly over half of the text is devoted to it, despite the fact that it is a somewhat neglected area by developmental biologists. A lot of what is known about fruit body formation is descriptive, although a few well-characterized aspects of the biology of these organisms have been developed recently. For example, the analysis of mating type genes, gene expression in homokaryotic and dikaryotic mycelia and the role of hydrophobins in the development of aerial structures have all advanced considerably in recent years and are well covered here. The author also shows how growth of the
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16 Bauer, M.W., Bauer, S.H. and Kelly, R.M. (1997) Appl. Environ. Microbiol. 63, 1160–1164 17 Cole, S.T. et al. (1998) Nature 393, 537–544 18 Knipfer, N. and Shrader, T.E. (1997) Mol. Microbiol. 25, 375–383 19 Nagy, I. et al. (1998) J. Bacteriol. 180, 5448–5453 20 Roudiak, S.G. et al. (1998) Biochemistry 37, 377–386 21 Wolf, S. et al. (1998) J. Mol. Biol. 277, 13–25 22 McCormack, T. et al. (1997) J. Biol. Chem. 272, 26103–26109 23 Seemüller, E., Lupas, A. and Baumeister, W. (1996) Nature 382, 468–470 24 Zühl, F. et al. (1997) FEBS Lett. 418, 189–194 25 Ruepp, A. et al. (1998) FEBS Lett. 425, 87–90 26 Kanemori, M. et al. (1997) J. Bacteriol. 179, 7219–7225 27 Fenteany, G. and Schreiber, S.L. (1998) J. Biol. Chem. 273, 8545–8548 28 Ichetovkin, I.E., Abramochkin, G. and Shrader, T.E. (1997) J. Biol. Chem. 272, 33009–33014 29 Keiler, K.C., Patrick, R.H. and Sauer, R.T. (1996) Science 271, 990–993 30 Gottesman, S. et al. (1998) Genes Dev. 12, 1338–1347 31 Herman, C. et al. (1998) Genes Dev. 12, 1348–1355 32 Löwe, J. et al. (1995) Science 268, 533–539 33 Groll, M. et al. (1997) Nature 386, 463–471
basidiocarp is regulated so that the spore-bearing gills are presented in optimal alignment for efficient liberation of spores into the environment. Material is brought together from a range of sources and approaches to provide a useful overview of the present status of the field as a whole and to highlight hot topics for the future. As for the rest of the book, I must confess to being a little disappointed about what is left out. The recent strides forward in our understanding of the regulation and molecular events concerned with cell polarity and bud site selection and the interplay between cell cycle regulation and bud morphogenesis (all in yeast) are not mentioned. The regulation of pseudohyphal and hyphal growth in yeasts is covered in only a paragraph, and recent molecular genetic analyses of hyphal growth, septum formation and branching in Aspergillus are barely touched on. These studies have pushed fungal morphogenesis to the forefront of developmental biology and have been reported in the most prestigious journals. To my mind, they are of profound importance to our appreciation of, for example, hyphal tip growth, branch for-
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mation and clamp connections in all fungi and, as such, I would have expected them to have been a major part of the book. Perhaps the omission of these key areas is the result of the strong focus on basidiomycete development. The book also has a whole chapter devoted to metabolism and biochemistry, with figures showing all the major metabolic pathways. Fine, but these pathways are generally common to most life forms and cannot be seen as essential to the theme of the book. Perhaps it was a tall order to get the broad coverage that I anticipated from the title. I can see this book being highly recommended for those working on, or wanting to know more about, the morphogenesis of higher fungi. However, those of a molecular genetic bent who are interested in a wider diversity of model fungal systems or the general molecular mechanisms underlying morphogenesis will feel less well served. Neil Gow Dept of Molecular and Cell Biology, Marischal College, University of Aberdeen, Aberdeen, UK AB25 2ZD.
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