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The TPR snap helix: a novel protein repeat motif from mitosis to transcription
TIBS 16-MAY1991
REVIEWS IN THE PAST FEW YEARS much progress has been made in understanding how the eukaryotic cell division cycle is controlled. The M-phase kinase, cyclin and protein phosphatase are among the universal regulators of mitosis in eukaryotic cells =. In yeasts, genes encoding these proteins were identified from mutations that cause cells to be defective at discrete steps of the cell cycle and were thus known as cell division cycle (cdc) genes2. Recently, a novel relationship was found among a group of mitotic genes 3 that includes budding yeast CDCI6, CDC23, fission yeast nuc2+ (nuclear alteration) and filamentous fungi BimA (block in mitosis). The product of each of these genes contains a characteristic 34-amino acid repeat that is defined by a degenerate consensus sequence. The same repeating motif has also been found in several proteins participating in RNA synthesis regulation, protein import and Drosophila development 3,4. It has been postulated that the repeats form jointed helical structures, each with a 'knob' and 'hole' such that the knob of one TPR helix can associate with the hole of a second TPR helixs.
TheTPR motif The re[beat motif was first noted through inspection of the predicted product of the CDC23gene3; a computer search detected similarities to five other genes: budding yeast CDCI6 (Ref. 6), SSN6 (Ref. 7) and SM3 (Ref. 8), fission yeast nuc2* (Ref. 9) and AsFergUlus BimA ~ . O'Donnel and R. Morris, pers. commun.). On closer inspection, the regions of similarity consisted of multiple copies of a 34amino acid motif which has been named the tetratricopeptide (TPR) repeat (Fig. la). The consensus repeat sequences are degenerate, but the spacing of each repeat is rather strict. M. Goeblis at the Departmentof Biochemistryand MolecularBiology,and the WaltherOncologyCenter~IndianaUniversity School of Medicine,635 BarnhillDrive, Indianapolis,IN 46202-5122, USA. M. Yanagldais at the Departmentof Biophysics, Facultyof Science,Kyoto University,Sakyo-ku,Kyoto606, Japan.
The TPR snap helix: a novel protein repeat n]o tif from n]itosis to tra nscrit)tion
The recently discovered TPR gene family encodes a diverse group of proteins that function in mitosis, transcription, splicing, protein import and neurogenesis. These multi
The number of identified repeats varies among the different proteins, ranging between 7 and 16 (Fig. Ib), and in most cases, the repeats are arranged as tandem arrays. A particular repeat in CDC23, nuc2"/BimA and CDC16/cut9* genes varies significantly from the other repeats. This repeat, designated 34v (Ref. 5), is present only in the genes required for mitosis.
the chromosomes condense and form a metaphase plate-like structure with a short spindle that fails to elongate. Addition of neurotubulin and ATP to the arrested spindle in vitro induced spindle elongation, but did not allow chromosome disjunction, suggesting that the defect lies in the chromatin structure rather than the spindle 13. The nuc2 ÷ protein is located in the nucleus,
TheTPRgenesand products Although the ~ o w n TPR proteins listed in Table ! have varied functions, many of them are associated with the nucleus. Mutations in CDCI6 and CDC23 cause arrest before entry into mitosis (G2/M) after DNA replication and increased levels of chromosome missegregation 2.=°. Abnormal nuclear DNA movements were observed in the mutant cells ==. The fission yeast mitotic gene cut9+ (Ref. 12) was recently cloned and found to be similar to CDCI6 (I. Samejima and M. Yanagida, unpublished). The cut9 ÷ protein is localized in the nucleus. The phenotype of the cut9 mutant resembled that of nuc2. The metaphase/anaphase transition of mitosis is blocked in the temperaturesensitive (ts) nuc2 mutantg:
© 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00
173
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Rgum 1 TPR proteins and their repeated sequences. (a) The repeated regions of five TPR proteins are shown. Conserved amino acids among different proteins are boxed. Conserved amino acids within the repeats of the same proteins are circled (b) Locations of the repeats in TPR proteins. Each box represents a repeat unit; the crossed one is imperfect in repeat unit length, the hatched box represents the 34 v repeat unit (see text) and the box with the dot contains the essential glycine5. 174
cofractionating with the nuclear matrix/scaffold fraction 9. This localization is supported by anti-nuc2 antibodies that stained the nuclear chromosomal region of S. pombe (S. Uzawa, unpublished; Fig. 2). A unique domain of nuc2 ÷ protein binds to AT-rich DNA in vitro5. The predicted amino acid sequence of BimA protein more closely resembles nuc2* (K. O'Donnel and R. Morris, pers. commun.), even outside the repeated regions, than any of the other TPR proteins. The budding yeast gene SSN6 encodes a protein that functions as a negative regulator of gene expression with a broad range of action and is required for normal growth, sporulation and mating7. A null allele of SSN6 results in constitutive expression of invertase and other glucose-repressible genes. The SSN6 protein is localized in the nucleus ~4. The amino-terminal third of SSN6 protein, which contains the TPR unit, is important for SSN6 function, although only four of the ten TPR repeats are essentiai; the carboxyl terminus is phosphorylated but is dispensable. The TPR repeats in the SKI3 and STII gene products are of various lengths 4 (see Fig. I b). In SKI3 ('super killer') mutant cells, increased amounts of dsRNA yeast killer toxin are produced, which is thought to be due to derepression of transcription of killer toxin 8. Fusion proteins containing parts of SKI3 and ~galactosidase are targeted to the nucleus, showing that SKI3 is a nuclear protein. The STII gene product is stress inducible, but has no homology to the hsp70 family of heat shock proteins ~s.The function of the STII protein is unknown. The PRP6 gene has been cloned and sequenced, and its product found to be repetitive 's. Although the repeat unit was initially termed PW, it resembles a TPR motif (Fig. la). The gene product, although not identified, is presumed to be involved in an early step of the premRNA splicing ~7 pathway and so it is most likely a nuclear protein. The amino-terminal domain of the MAS70 protein is anchored to the outer mitochondria! membrane ~s. All of the TPR repeats are contained in the remaining cytoplasmic domain. MAS70 appears to accelerate the import of mitochondrial proteins. Mutant crn (Drosophila crooked neck) embryos display defects late in emb"vogenesis, primarily in those lineages of the nervous system. The crn gene is transcribed in all cells during embryonic developme,!
TIBS 16-MAY1991
and the mutant phenotype suggests that crn may be required for cell division. The predicted crn gene product contains 16 TPR motifs, though four of them are much less conserved (D. Smouse, K. Zhang and N. Perrimon, pets. commun.). The TPR motif is essential Although most of the TPR proteins have been identified through mutations in lower eukaryotes or Drosophila, only for nuc2 is the exact mutation site known. The mutation within the nuc2663 allele resides in one of the TPR repeatss. A single amino acid substitution (GlyS04->Asp) leads to the phenotypes of mitotic arrest, sterility and caffeine hypersensitivity. This Gly residue is conserved in one of the TPR repeat~ of CDC23, nuc2*/BimA, CDCl6/cut9*, SSN6 and SM3 (Fig. lb). Truncated nuc2 polypeptides that are missing the terminal TPR repeats did not complement the temperature-sensitive mutant, indicating that most of the TPR repeats are necessary for the function of nuc2* protein, in the ninth TPR repeat of the cut9 mutant allele a conserved Ala is replaced by Thr (I. Samejima and M. Yanagida, unpublished). Single amino acid substitutions in the TPR domain of CDC23 also caused a temperature-sensitive phenotype (R. Sikorski and R Hieter, pers. commun.). The TPR domain of MAS70 mitochondrial protein is exposed to the cytoplasm and has been shown to be necessary for the function of the protein, but not for its own targeting to the mitochondrion tg. Structural models A recombinant, truncated nuc2 protein, containing nine TPR repeats was purified for analysis s. Circular dichroism (CD) studies showed that the TPR repeats were approximately 50% a-helical with no [i-sheet structure. A ladder of polypeptide bands with an interval of 3-4 kDa (roughly equivalent to one TPR repeat) was produced by limited proteolysis; a similar result was obtained using native nuc2 ÷ protein. Electron microscopy showed that the TPR repeats could be assembled into filamentous structures. Thus recombinant truncated nuc2 protein appe~s to form a helix-containing str~cture that is protease-sensitive at 3--4 kDa intervals. The TPR consensus sequence can be divided into two subdomains, A and B (Fig. 3a), that are stereochemically c o m -
plementary in the a-helical configuration, forming a 'knob' and 'hole' (Fig. 3b; Ref. 5). The two most conserved in the TPR repeats are of central importance: Gly (or Ala) has a small side chain and forms the hole, while the bulky Tyr (or Phe) serves as the knob. These residues are well placed to interact between adjacent helices of the correct orientation. Secondary structure prediction of the TPR repeats 3 also supports such Figure 2 an amphipathic helical Nuclear location of nuc2* protein. Ruorescencelight micrographof a fission yeast cell stained by anti-nuc2 model. antibodies (left) and the same cell by a DNA-specific The amino acid residues fluorescent dye, DAPI (dght). A mitotically arrested surrounding the knob and nda3 (lS-tubulin)mutant was employed. The bar indihole are hydrophobic and cates 1 pm. probably function to stabilize the association of the two helices. The helix-breaking Pro the TPR motif also exist in higher residue is present at the carboxyl end eukaryotes? As most of these proteins of the repeat, which is consistent with were predicted from DNA sequence analysis of genes cloned by the compthe protease-sensitive hinge or turn. A plausible model for nuc2 ÷ protein lementation of mutant phenotypes, it is is illustrated in Fig. 3c. The putative not surprising that no man'.,malianmemDNA-binding region, which is highly bers have been found. Low cellular conhydrophilic, is looped out from the centrations (e.g. only 2000 copies per compactly folded, repeated regions. A cell for nuc2" protein; Ref. 9) may also possible involvement of the repeated hamper their detection. The PCR (polstructure for the association of chroma- ymerase chain reaction) method may tid DNA was discussed elsewhere 2°. In be of use in finding higher eukaryotic the other members of TPR proteins, the homologs. A search for mammalian unique regions may serve as the signal homologs of CDC16/cut9" and nuc2*/ for binding to transcriptional factors or BimA is now in progress. Nuclear matrix/scaffold structures spliceosomes or anchoring the protein are thought to play important roles in at mitochondrial outer membrane. chromosome structure, DNA replication and gene expression. Their protein TPR motifs, matdx structure and cell components are insoluble at high salt replation With the exception of the Drosophila concentration (2 M NaCI) or in detercrn protein, all of the known TPR pro- gents [2% Nonidet or 25mM lithium teins (Table I) are of fungal origin. Does iodo-salicylate (LIS)] and form a netTable I. TPR genes, products,cell location and function Genes
Organisms
CDC16 CDC23 SSN6 SKI3 STI1 PRP6 MASTO nuc2+ cut9 + BirnA crn
S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. pornbe S. pombe A. nidulans Drosophila
Numberof amino acids 840 626 966 1423 589 899 617 665 671
702
Numberoff Cell location repeats 10 9 10 8 8 9 7 10 10 10 16
Nucleus ND Nucleus Nucleus ND Nucleus Mitochondria Nucleus Nucleus ND ND
Function
Refs
mitosis mitosis transcription transcription stress induced splicing protein import mitosis mitosis mitosis neurogenesis
6,d 3 7 8 14 16 17 9 a b c
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175
TIBS 1 6 - MAY1991
have a similar association. While some are localized in the nucleus, others are related to nuclear functions such as chromosome segregation, transcriptional regulation and splicing. Only future work will clarify the structural and func-
work-like structure in the mammalian nucleus (for review see Ref. 21). The fission yeast nuc2 ÷ protein was shown to be a component of the nuclear matrix fraction, it remains to be seen whether other TPR members will also
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Rgure 3 The snap helix with knoL and hole. (a) An or-helical net with 3.6 residues per turn is depicted for one 34-amino acid (TPR) repeat. Amino acids of the unified consensus sequence are clustered in the two regions, A and B. The asterisks indicate large hydrophobic amino acids (L, I, V, F, Y and W). The conserved glycine is found within A, and B contains the conserved tyrosine. (b) A computer graphic image of association between two regions A and B of different helices by the knob-into-hole binding motif. In this case, two helices run antiparallel to one another. Dots represent the van der Waals radii. (¢) A schematic illustration for nuc2* protein. The TPR segments may form a tightly packed helix-turn domain from which the DNA-binding domain protrudes. The putative knob-into-hole association is tinted. 176
tional relationships between nuclear TPR proteins and the nuclear matrix] scaffold. How can the role of the mitochondrial MAS70 protein be reconciled with that of the other TPR proteins? The TPR repeats of MAS70 may interact with cytoplasmic matrices that may contain constituents related to those of the nuclear matrix. Alternatively, other cytoplasmic TPR proteins might remain to be discovered. Do the TPR repeats in different proteins share any common function? Structural features of the postulated snap helix strongly suggest that association of the helical segments is stereochemically feasible, with hydrophobic re~,idues to stabilize the association. Factors influencing these associations should be able to alter the biological activity of the TPR proteins. An amino acid substitution in the sixth TPR repeat of nuc2-663 causes the mutant protein to remain soluble as well as to migrate with a reduced electrophoretic mobility9. Therefore, ligands affecting the conformation of TPR repeats may regulate the activity of TPR proteins with subtle effects on these ligands possibly being amplified by the TPR repeats. To explain the mutant phenotypes, we postulate that the cellular concentration of such a iigand (protein or low molecular weight substance) may be inducible or cell-cycle regulated. TPR proteins may accept certain cellular signals or transmit them to other cellular components. Therefore, it is intriguing that many of the genes encoding TPR proteins have been found to be functionally related to members of another family of proteins that are characterized by the presence of a different internally repetitive domain, comprising repeats of a 43-amino acid unit, first described within the [I-subunit of the heterotrimeric G protein transducin 22. Genetic associations have been described for PRP6/PRP4 (Ref. 23), SSN6/TUP1 (Ref. 24), CDCI6, CDC23/ CDC20 (D. Burke, pets. commun.), and possibly SKI3/MAKI1 (Ref. 25); each pair of proteins consists of a TPR protein and ~transducin-related prot?in, respectively.
Acknowledgements We are greatly indebted to our col-
leagues for communicating unpublished information to us, particularly to D. Burke, P. Hieter, D. Koshland, R. Morris, G. Schatz and D. Smouse. We thank
TIBS16-MAY1991 S. Uzawa for the immunofluorescence micrograph, T. Hirano for computer graphics and l. Samejima for figure preparation. References I Lewin, 8. (1990) Cell61, 743-752 2 Pringle, J. and Hartwell, L. (1981) The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance (Strathern, J. N., Jones, E. W. and Broach, J. R., eds), pp. 97-142, Cold Spring Harbor Press 3 Sikorski, R. S., Boguski, M. S., Goebl, M. and Hieter, P. (1990) Ce/160, 307-317 4 Boguski, M. S., Sikorski, R. S., Hieter, P. and Goebl, M. (1990) Nature 346, 114 5 Hirano, T., Kinoshita, N., Morikawa, K. and Yanagida, M. (1990) Cell60, 319-328
6 Icho, T. and Wickner, R. B. (1987) Nucleic Acids Res. 15, 8439-8450 7 Schultz, J. and Carlson, M. (1987) Mol. Cell. Biol. 7, 3637-3645 8 Rhee, S. K., Icho, T. and Wickner, R. B. (1989) Yeast 5,149-158 9 Hirano, T., Hiraoka, Yoand Yanagida, M. (1988) J. Cell BioL 106,1171-1183 10 Hartwell, L. H. and Smith, D. (1985) Genetics 110, 381-395 11 Palmer, R. E., Koval, M. and Koshland, D. (1989) J. Cell Biol. 109, 3355-3366 12 Hirano, T., Funahashi, S., Uemura, T. and Yanagida, M. (1986) EMBO J. 5, 2973-2979 13 Masuda, H., Hirano, T., Yanagida, M. and Cande, W. Z. (1990) J. Cell Biol. 110, 417-425 14 Schultz, J., Marshall-Carlson, L. and Carlson, M. (1990) Mol. Cell. BioL 10, 4744-4756 15 Nicolet, C. M. and Craig, E. A. (1989) MoL Cell. Biol. 9, 3638-3646 16 Legrain, P. and Chou|ika, A. (1990) EMBO J. 9,
2775--2781 17 Woolford, J. L., Jr (1989) Yeast 5, 439-457 18 Hase, T., Riezman, H., Suda, K. and Schatz, G. (1983) EMBO J. 2, 2169-2172 19 Riezman, H., Hase, T., van Loon, A., Grivell, L. A., Suda, K. and Schatz, G. (1983) EMBO J. 2, 2161-2168 20 Yanagida, M. (1990) J. Cell Sci. 96,1-3 21 Gasser, S. M., Laroche, T., Falquet, J., Tour, E. B. and Laemmli, U. K. (1986) J. MoL BioL 188, 613-629 22 Fong, H. K. W., Hurley, J. B., Hopkins, R. S., Miake-Lye, R., Johnson, M. S., Doolittle, R. F. and Simon, M. I. (1986) Proc. Natl Acad. Sci. USA 83, 2162-2166 23 Dalrymple, M. A., Petersen-Bjorn, S., Fiesen, J. D. and Beggs, J. D. (1990) Cell58, 811-812 24 Williams, F. E. and Tumbly, R. J. (1990) Mol. Cell. Biol. 10, 6500-6511 25 Icho, T. and Wickner, R. B. (1988) J. Biol. Chem. 263, 1467-1475
A common denomifffi[-6 i: linking GLYCOGEN METABOLISM may have negative connotations for many, especially students hard-pressed to remember the difference between phosphorylase, phosphorylation and phosphatase. However, many important insights into cellular regulation have emerged from this field, which provided the first examples of an enzyme regulated by phosphorylation (glycogen phosphorylase), second messengers (cyclic AMP) and regulatory cascades containing multiple protein kinases, to name but a few. Recent evidence now links the regulation of the nuclear protooncogenes c-Jun and c-Myb, a Drosophila developmental gene and a protein kinase first described in the context of glycogen metabolism.
A bdof bbtoff of glycogensynthase klnaN-3 • Following the elucidation of the mechanism of action of adrenaline on glycogen breakdown, in the late 1970s attention turned to the molecular basis underlying promotion of glycogen deposition by insulin. In this system, insulin activates the rate-limiting enzyme of glycogen synthesis, glycogen synthase, by reducing its phosphate contentL However, in contrast to the enzyme degrading glycogen, phosphorylase, which is targeted by a single protein kinase (phosphorylase kinase), it soon became clear that glycogen synthase was phosphorylated by multiple protein kinases at multiple sites (for review J. R. Woodgettis at the LudwigInstitutefor CancerResearch,91 RidingHouseStreet, LondonWIP 8BT,UK.
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A ubiquitous protein-serine kinase, initially implicated in glycogen regulation, has surfaced unexpectedly in the fields of nuclear oncogenes and fruitfly development. This unusual linkage may reflect the role of this kinase in phosphorylating proteins normally activated by dephosphorylation, thus providing a priming function. Loss of such a primer would result in constitutive activation of substrates, a scenario concordant with the dramatic and pleiotropic phenotype observed in Drosophila null mutants.
see Ref. 2). Perhaps if workers then had known that the tally was to be a minimum of eight protein kinases acting on at least nine sites, they might have been daunted by the task ahead! Initially, four groups reported the resolution and partial purification of some five glycogen synthase kinases. In the terminology of Cohen, glycogen synthase kinase-I (GSK-I) equates to cyclic AMP-dependent protein kinase, GSK-2 is phosphoryla~e kinase and GSK-5 is casein kinase il. GSK-3 and GSK-4 were, at the time, novel enzymes of unknown function. GSK-3 phosphorylated four clustered serine residues in glycogen synthase, which resulted in dramatic inactivation of the enzyme2'3. Furthermore, analysis of the phosphate content of the individual sites on glycogen synthase following insulin treatment
© 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00
revealed the largest change to be a reduction in the sites targeted by GSK-3 (Ref. I). Inhibition of GSK-3 would thus mimic the effect of insulin, potentially implicating this protein-serine kinase in insulin action. Equally tenable of course would be insulin activation of a protein phosphatase (see below). While most attention was focused on the role of protein kinases in hormonal regulation, the possible involvement of protein phosphatases was also being pursued. In particular, the group of Merlevede described the existence of a dormant protein phosphatase (termed Fo a form of protein phosphatase-l) that became active in the presence of MgATP and a protein fraction termed factor A (FA)4. In addition to activating Fo this protein ~.xhibited glycogen synthase kinase acti~;ity and it 177
REVIEWS IN THE PAST FEW YEARS much progress has been made in understanding how the eukaryotic cell division cycle is controlled. The M-phase kinase, cyclin and protein phosphatase are among the universal regulators of mitosis in eukaryotic cells =. In yeasts, genes encoding these proteins were identified from mutations that cause cells to be defective at discrete steps of the cell cycle and were thus known as cell division cycle (cdc) genes2. Recently, a novel relationship was found among a group of mitotic genes 3 that includes budding yeast CDCI6, CDC23, fission yeast nuc2+ (nuclear alteration) and filamentous fungi BimA (block in mitosis). The product of each of these genes contains a characteristic 34-amino acid repeat that is defined by a degenerate consensus sequence. The same repeating motif has also been found in several proteins participating in RNA synthesis regulation, protein import and Drosophila development 3,4. It has been postulated that the repeats form jointed helical structures, each with a 'knob' and 'hole' such that the knob of one TPR helix can associate with the hole of a second TPR helixs.
TheTPR motif The re[beat motif was first noted through inspection of the predicted product of the CDC23gene3; a computer search detected similarities to five other genes: budding yeast CDCI6 (Ref. 6), SSN6 (Ref. 7) and SM3 (Ref. 8), fission yeast nuc2* (Ref. 9) and AsFergUlus BimA ~ . O'Donnel and R. Morris, pers. commun.). On closer inspection, the regions of similarity consisted of multiple copies of a 34amino acid motif which has been named the tetratricopeptide (TPR) repeat (Fig. la). The consensus repeat sequences are degenerate, but the spacing of each repeat is rather strict. M. Goeblis at the Departmentof Biochemistryand MolecularBiology,and the WaltherOncologyCenter~IndianaUniversity School of Medicine,635 BarnhillDrive, Indianapolis,IN 46202-5122, USA. M. Yanagldais at the Departmentof Biophysics, Facultyof Science,Kyoto University,Sakyo-ku,Kyoto606, Japan.
The TPR snap helix: a novel protein repeat n]o tif from n]itosis to tra nscrit)tion
The recently discovered TPR gene family encodes a diverse group of proteins that function in mitosis, transcription, splicing, protein import and neurogenesis. These multi
The number of identified repeats varies among the different proteins, ranging between 7 and 16 (Fig. Ib), and in most cases, the repeats are arranged as tandem arrays. A particular repeat in CDC23, nuc2"/BimA and CDC16/cut9* genes varies significantly from the other repeats. This repeat, designated 34v (Ref. 5), is present only in the genes required for mitosis.
the chromosomes condense and form a metaphase plate-like structure with a short spindle that fails to elongate. Addition of neurotubulin and ATP to the arrested spindle in vitro induced spindle elongation, but did not allow chromosome disjunction, suggesting that the defect lies in the chromatin structure rather than the spindle 13. The nuc2 ÷ protein is located in the nucleus,
TheTPRgenesand products Although the ~ o w n TPR proteins listed in Table ! have varied functions, many of them are associated with the nucleus. Mutations in CDCI6 and CDC23 cause arrest before entry into mitosis (G2/M) after DNA replication and increased levels of chromosome missegregation 2.=°. Abnormal nuclear DNA movements were observed in the mutant cells ==. The fission yeast mitotic gene cut9+ (Ref. 12) was recently cloned and found to be similar to CDCI6 (I. Samejima and M. Yanagida, unpublished). The cut9 ÷ protein is localized in the nucleus. The phenotype of the cut9 mutant resembled that of nuc2. The metaphase/anaphase transition of mitosis is blocked in the temperaturesensitive (ts) nuc2 mutantg:
© 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00
173
TIBS 1 6 - M A Y 1 9 9 1
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Rgum 1 TPR proteins and their repeated sequences. (a) The repeated regions of five TPR proteins are shown. Conserved amino acids among different proteins are boxed. Conserved amino acids within the repeats of the same proteins are circled (b) Locations of the repeats in TPR proteins. Each box represents a repeat unit; the crossed one is imperfect in repeat unit length, the hatched box represents the 34 v repeat unit (see text) and the box with the dot contains the essential glycine5. 174
cofractionating with the nuclear matrix/scaffold fraction 9. This localization is supported by anti-nuc2 antibodies that stained the nuclear chromosomal region of S. pombe (S. Uzawa, unpublished; Fig. 2). A unique domain of nuc2 ÷ protein binds to AT-rich DNA in vitro5. The predicted amino acid sequence of BimA protein more closely resembles nuc2* (K. O'Donnel and R. Morris, pers. commun.), even outside the repeated regions, than any of the other TPR proteins. The budding yeast gene SSN6 encodes a protein that functions as a negative regulator of gene expression with a broad range of action and is required for normal growth, sporulation and mating7. A null allele of SSN6 results in constitutive expression of invertase and other glucose-repressible genes. The SSN6 protein is localized in the nucleus ~4. The amino-terminal third of SSN6 protein, which contains the TPR unit, is important for SSN6 function, although only four of the ten TPR repeats are essentiai; the carboxyl terminus is phosphorylated but is dispensable. The TPR repeats in the SKI3 and STII gene products are of various lengths 4 (see Fig. I b). In SKI3 ('super killer') mutant cells, increased amounts of dsRNA yeast killer toxin are produced, which is thought to be due to derepression of transcription of killer toxin 8. Fusion proteins containing parts of SKI3 and ~galactosidase are targeted to the nucleus, showing that SKI3 is a nuclear protein. The STII gene product is stress inducible, but has no homology to the hsp70 family of heat shock proteins ~s.The function of the STII protein is unknown. The PRP6 gene has been cloned and sequenced, and its product found to be repetitive 's. Although the repeat unit was initially termed PW, it resembles a TPR motif (Fig. la). The gene product, although not identified, is presumed to be involved in an early step of the premRNA splicing ~7 pathway and so it is most likely a nuclear protein. The amino-terminal domain of the MAS70 protein is anchored to the outer mitochondria! membrane ~s. All of the TPR repeats are contained in the remaining cytoplasmic domain. MAS70 appears to accelerate the import of mitochondrial proteins. Mutant crn (Drosophila crooked neck) embryos display defects late in emb"vogenesis, primarily in those lineages of the nervous system. The crn gene is transcribed in all cells during embryonic developme,!
TIBS 16-MAY1991
and the mutant phenotype suggests that crn may be required for cell division. The predicted crn gene product contains 16 TPR motifs, though four of them are much less conserved (D. Smouse, K. Zhang and N. Perrimon, pets. commun.). The TPR motif is essential Although most of the TPR proteins have been identified through mutations in lower eukaryotes or Drosophila, only for nuc2 is the exact mutation site known. The mutation within the nuc2663 allele resides in one of the TPR repeatss. A single amino acid substitution (GlyS04->Asp) leads to the phenotypes of mitotic arrest, sterility and caffeine hypersensitivity. This Gly residue is conserved in one of the TPR repeat~ of CDC23, nuc2*/BimA, CDCl6/cut9*, SSN6 and SM3 (Fig. lb). Truncated nuc2 polypeptides that are missing the terminal TPR repeats did not complement the temperature-sensitive mutant, indicating that most of the TPR repeats are necessary for the function of nuc2* protein, in the ninth TPR repeat of the cut9 mutant allele a conserved Ala is replaced by Thr (I. Samejima and M. Yanagida, unpublished). Single amino acid substitutions in the TPR domain of CDC23 also caused a temperature-sensitive phenotype (R. Sikorski and R Hieter, pers. commun.). The TPR domain of MAS70 mitochondrial protein is exposed to the cytoplasm and has been shown to be necessary for the function of the protein, but not for its own targeting to the mitochondrion tg. Structural models A recombinant, truncated nuc2 protein, containing nine TPR repeats was purified for analysis s. Circular dichroism (CD) studies showed that the TPR repeats were approximately 50% a-helical with no [i-sheet structure. A ladder of polypeptide bands with an interval of 3-4 kDa (roughly equivalent to one TPR repeat) was produced by limited proteolysis; a similar result was obtained using native nuc2 ÷ protein. Electron microscopy showed that the TPR repeats could be assembled into filamentous structures. Thus recombinant truncated nuc2 protein appe~s to form a helix-containing str~cture that is protease-sensitive at 3--4 kDa intervals. The TPR consensus sequence can be divided into two subdomains, A and B (Fig. 3a), that are stereochemically c o m -
plementary in the a-helical configuration, forming a 'knob' and 'hole' (Fig. 3b; Ref. 5). The two most conserved in the TPR repeats are of central importance: Gly (or Ala) has a small side chain and forms the hole, while the bulky Tyr (or Phe) serves as the knob. These residues are well placed to interact between adjacent helices of the correct orientation. Secondary structure prediction of the TPR repeats 3 also supports such Figure 2 an amphipathic helical Nuclear location of nuc2* protein. Ruorescencelight micrographof a fission yeast cell stained by anti-nuc2 model. antibodies (left) and the same cell by a DNA-specific The amino acid residues fluorescent dye, DAPI (dght). A mitotically arrested surrounding the knob and nda3 (lS-tubulin)mutant was employed. The bar indihole are hydrophobic and cates 1 pm. probably function to stabilize the association of the two helices. The helix-breaking Pro the TPR motif also exist in higher residue is present at the carboxyl end eukaryotes? As most of these proteins of the repeat, which is consistent with were predicted from DNA sequence analysis of genes cloned by the compthe protease-sensitive hinge or turn. A plausible model for nuc2 ÷ protein lementation of mutant phenotypes, it is is illustrated in Fig. 3c. The putative not surprising that no man'.,malianmemDNA-binding region, which is highly bers have been found. Low cellular conhydrophilic, is looped out from the centrations (e.g. only 2000 copies per compactly folded, repeated regions. A cell for nuc2" protein; Ref. 9) may also possible involvement of the repeated hamper their detection. The PCR (polstructure for the association of chroma- ymerase chain reaction) method may tid DNA was discussed elsewhere 2°. In be of use in finding higher eukaryotic the other members of TPR proteins, the homologs. A search for mammalian unique regions may serve as the signal homologs of CDC16/cut9" and nuc2*/ for binding to transcriptional factors or BimA is now in progress. Nuclear matrix/scaffold structures spliceosomes or anchoring the protein are thought to play important roles in at mitochondrial outer membrane. chromosome structure, DNA replication and gene expression. Their protein TPR motifs, matdx structure and cell components are insoluble at high salt replation With the exception of the Drosophila concentration (2 M NaCI) or in detercrn protein, all of the known TPR pro- gents [2% Nonidet or 25mM lithium teins (Table I) are of fungal origin. Does iodo-salicylate (LIS)] and form a netTable I. TPR genes, products,cell location and function Genes
Organisms
CDC16 CDC23 SSN6 SKI3 STI1 PRP6 MASTO nuc2+ cut9 + BirnA crn
S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. pornbe S. pombe A. nidulans Drosophila
Numberof amino acids 840 626 966 1423 589 899 617 665 671
702
Numberoff Cell location repeats 10 9 10 8 8 9 7 10 10 10 16
Nucleus ND Nucleus Nucleus ND Nucleus Mitochondria Nucleus Nucleus ND ND
Function
Refs
mitosis mitosis transcription transcription stress induced splicing protein import mitosis mitosis mitosis neurogenesis
6,d 3 7 8 14 16 17 9 a b c
ND, not determined. a I. Samejima and M. v ~ (unpublished); b K. O'Donnel and R. Morris (gem. commun.); ¢ D. Smouse, I~ Zang and N. Perrimon (gem. commun.); d R. E. Palmer and D. Koshland (gem. commun.).
175
TIBS 1 6 - MAY1991
have a similar association. While some are localized in the nucleus, others are related to nuclear functions such as chromosome segregation, transcriptional regulation and splicing. Only future work will clarify the structural and func-
work-like structure in the mammalian nucleus (for review see Ref. 21). The fission yeast nuc2 ÷ protein was shown to be a component of the nuclear matrix fraction, it remains to be seen whether other TPR members will also
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tional relationships between nuclear TPR proteins and the nuclear matrix] scaffold. How can the role of the mitochondrial MAS70 protein be reconciled with that of the other TPR proteins? The TPR repeats of MAS70 may interact with cytoplasmic matrices that may contain constituents related to those of the nuclear matrix. Alternatively, other cytoplasmic TPR proteins might remain to be discovered. Do the TPR repeats in different proteins share any common function? Structural features of the postulated snap helix strongly suggest that association of the helical segments is stereochemically feasible, with hydrophobic re~,idues to stabilize the association. Factors influencing these associations should be able to alter the biological activity of the TPR proteins. An amino acid substitution in the sixth TPR repeat of nuc2-663 causes the mutant protein to remain soluble as well as to migrate with a reduced electrophoretic mobility9. Therefore, ligands affecting the conformation of TPR repeats may regulate the activity of TPR proteins with subtle effects on these ligands possibly being amplified by the TPR repeats. To explain the mutant phenotypes, we postulate that the cellular concentration of such a iigand (protein or low molecular weight substance) may be inducible or cell-cycle regulated. TPR proteins may accept certain cellular signals or transmit them to other cellular components. Therefore, it is intriguing that many of the genes encoding TPR proteins have been found to be functionally related to members of another family of proteins that are characterized by the presence of a different internally repetitive domain, comprising repeats of a 43-amino acid unit, first described within the [I-subunit of the heterotrimeric G protein transducin 22. Genetic associations have been described for PRP6/PRP4 (Ref. 23), SSN6/TUP1 (Ref. 24), CDCI6, CDC23/ CDC20 (D. Burke, pets. commun.), and possibly SKI3/MAKI1 (Ref. 25); each pair of proteins consists of a TPR protein and ~transducin-related prot?in, respectively.
Acknowledgements We are greatly indebted to our col-
leagues for communicating unpublished information to us, particularly to D. Burke, P. Hieter, D. Koshland, R. Morris, G. Schatz and D. Smouse. We thank
TIBS16-MAY1991 S. Uzawa for the immunofluorescence micrograph, T. Hirano for computer graphics and l. Samejima for figure preparation. References I Lewin, 8. (1990) Cell61, 743-752 2 Pringle, J. and Hartwell, L. (1981) The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance (Strathern, J. N., Jones, E. W. and Broach, J. R., eds), pp. 97-142, Cold Spring Harbor Press 3 Sikorski, R. S., Boguski, M. S., Goebl, M. and Hieter, P. (1990) Ce/160, 307-317 4 Boguski, M. S., Sikorski, R. S., Hieter, P. and Goebl, M. (1990) Nature 346, 114 5 Hirano, T., Kinoshita, N., Morikawa, K. and Yanagida, M. (1990) Cell60, 319-328
6 Icho, T. and Wickner, R. B. (1987) Nucleic Acids Res. 15, 8439-8450 7 Schultz, J. and Carlson, M. (1987) Mol. Cell. Biol. 7, 3637-3645 8 Rhee, S. K., Icho, T. and Wickner, R. B. (1989) Yeast 5,149-158 9 Hirano, T., Hiraoka, Yoand Yanagida, M. (1988) J. Cell BioL 106,1171-1183 10 Hartwell, L. H. and Smith, D. (1985) Genetics 110, 381-395 11 Palmer, R. E., Koval, M. and Koshland, D. (1989) J. Cell Biol. 109, 3355-3366 12 Hirano, T., Funahashi, S., Uemura, T. and Yanagida, M. (1986) EMBO J. 5, 2973-2979 13 Masuda, H., Hirano, T., Yanagida, M. and Cande, W. Z. (1990) J. Cell Biol. 110, 417-425 14 Schultz, J., Marshall-Carlson, L. and Carlson, M. (1990) Mol. Cell. BioL 10, 4744-4756 15 Nicolet, C. M. and Craig, E. A. (1989) MoL Cell. Biol. 9, 3638-3646 16 Legrain, P. and Chou|ika, A. (1990) EMBO J. 9,
2775--2781 17 Woolford, J. L., Jr (1989) Yeast 5, 439-457 18 Hase, T., Riezman, H., Suda, K. and Schatz, G. (1983) EMBO J. 2, 2169-2172 19 Riezman, H., Hase, T., van Loon, A., Grivell, L. A., Suda, K. and Schatz, G. (1983) EMBO J. 2, 2161-2168 20 Yanagida, M. (1990) J. Cell Sci. 96,1-3 21 Gasser, S. M., Laroche, T., Falquet, J., Tour, E. B. and Laemmli, U. K. (1986) J. MoL BioL 188, 613-629 22 Fong, H. K. W., Hurley, J. B., Hopkins, R. S., Miake-Lye, R., Johnson, M. S., Doolittle, R. F. and Simon, M. I. (1986) Proc. Natl Acad. Sci. USA 83, 2162-2166 23 Dalrymple, M. A., Petersen-Bjorn, S., Fiesen, J. D. and Beggs, J. D. (1990) Cell58, 811-812 24 Williams, F. E. and Tumbly, R. J. (1990) Mol. Cell. Biol. 10, 6500-6511 25 Icho, T. and Wickner, R. B. (1988) J. Biol. Chem. 263, 1467-1475
A common denomifffi[-6 i: linking GLYCOGEN METABOLISM may have negative connotations for many, especially students hard-pressed to remember the difference between phosphorylase, phosphorylation and phosphatase. However, many important insights into cellular regulation have emerged from this field, which provided the first examples of an enzyme regulated by phosphorylation (glycogen phosphorylase), second messengers (cyclic AMP) and regulatory cascades containing multiple protein kinases, to name but a few. Recent evidence now links the regulation of the nuclear protooncogenes c-Jun and c-Myb, a Drosophila developmental gene and a protein kinase first described in the context of glycogen metabolism.
A bdof bbtoff of glycogensynthase klnaN-3 • Following the elucidation of the mechanism of action of adrenaline on glycogen breakdown, in the late 1970s attention turned to the molecular basis underlying promotion of glycogen deposition by insulin. In this system, insulin activates the rate-limiting enzyme of glycogen synthesis, glycogen synthase, by reducing its phosphate contentL However, in contrast to the enzyme degrading glycogen, phosphorylase, which is targeted by a single protein kinase (phosphorylase kinase), it soon became clear that glycogen synthase was phosphorylated by multiple protein kinases at multiple sites (for review J. R. Woodgettis at the LudwigInstitutefor CancerResearch,91 RidingHouseStreet, LondonWIP 8BT,UK.
0
lycogen metabolism ' nuclear
........
oncogenes and development
A ubiquitous protein-serine kinase, initially implicated in glycogen regulation, has surfaced unexpectedly in the fields of nuclear oncogenes and fruitfly development. This unusual linkage may reflect the role of this kinase in phosphorylating proteins normally activated by dephosphorylation, thus providing a priming function. Loss of such a primer would result in constitutive activation of substrates, a scenario concordant with the dramatic and pleiotropic phenotype observed in Drosophila null mutants.
see Ref. 2). Perhaps if workers then had known that the tally was to be a minimum of eight protein kinases acting on at least nine sites, they might have been daunted by the task ahead! Initially, four groups reported the resolution and partial purification of some five glycogen synthase kinases. In the terminology of Cohen, glycogen synthase kinase-I (GSK-I) equates to cyclic AMP-dependent protein kinase, GSK-2 is phosphoryla~e kinase and GSK-5 is casein kinase il. GSK-3 and GSK-4 were, at the time, novel enzymes of unknown function. GSK-3 phosphorylated four clustered serine residues in glycogen synthase, which resulted in dramatic inactivation of the enzyme2'3. Furthermore, analysis of the phosphate content of the individual sites on glycogen synthase following insulin treatment
© 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00
revealed the largest change to be a reduction in the sites targeted by GSK-3 (Ref. I). Inhibition of GSK-3 would thus mimic the effect of insulin, potentially implicating this protein-serine kinase in insulin action. Equally tenable of course would be insulin activation of a protein phosphatase (see below). While most attention was focused on the role of protein kinases in hormonal regulation, the possible involvement of protein phosphatases was also being pursued. In particular, the group of Merlevede described the existence of a dormant protein phosphatase (termed Fo a form of protein phosphatase-l) that became active in the presence of MgATP and a protein fraction termed factor A (FA)4. In addition to activating Fo this protein ~.xhibited glycogen synthase kinase acti~;ity and it 177