Fasshauer, D.

Jahn, R.

English

MPG.PuRe

360 VOLUME 14   NUMBER 5   MAY 2007   NATURE STRUCTURAL & MOLECULAR BIOLOGYBudding insights on cell polarityDirk Fasshauer & Reinhard JahnThe yeast proteins Sro7 and Sro77, and their metazoan relatives Lgl and tomosyn, interact with plasma membrane SNARE proteins and control cell polarity. Recently, the crystal structure of yeast Sro7 has been solved, yielding fascinating insights and even more unanswered questions concerning the molecular mechanism of these proteins.Sro proteins are large soluble proteins with close relatives in all eukaryotes. They  contain multiple WD40 repeats of ~40 amino acid  residues, typically containing a Trp-Asp pair at the carboxyl end. WD40 repeats are known to form blade-like  structures that circularly assemble into β-propellers. A metazoan  relative of Sro  proteins, the tumor  suppressor lethal giant larvae (Lgl), is important in  establishing  epithelial cell polarity during  embryogenesis in Drosophila melanogaster 8,9. The  ensitivity to NaCl of a yeast Sro  double mutant is  rescued by  ectopic  expression of the Drosophila lgl gene10. A mammalian  ortholog of Lgl has been shown to  interact with  syntaxin-4 (ref. 11), a  ubiquitous Qa-SNARE of the plasma  membrane. The  closest  relative of Sro in  animals is tomosyn,  originally  isolated as a binding partner of the Qa-SNARE syntaxin-1 (ref. 12). Syntaxin-1  mediates  neuronal  exocytosis, together with its SNARE  partners  synaptobrevin-2 (R) and SNAP-25 (Qbc). Thus, it appears that Sro, Lgl and  tomosyn carry out similar functions by  interacting with plasma membrane– specific Q-SNAREs.The new crystal structure of Sro7 (ref. 5) shows that the 14 WD40 repeats fold into two  consecutive seven-bladed β- propeller domains (Fig. 1a,b). The N-terminal  portion of the chain associates with the C- terminal β- propeller to form a molecular ‘clasp’,  yielding an  overall  topology similar to the actin- interacting  protein Aip1 (also called Unc78)13,14. The  arrangement of the two β-propellers into an open clamshell-shaped  structure provides  multiple protein- binding surfaces oriented at specific angles with respect to one another. One of these  surfaces is  occupied by the 60-residue C- terminal tail of Sro7 (residues 892–951), which has an extended  conformation that tightly packs against the convex surface of the N- terminal β-propeller. A comparable  arrangement has been found for the seven-bladed β- propeller proteins coronin-1 and Nup214 (also called CAN). In coronin-1, the tight binding of a 130-residue C- terminal tail stabilizes the β-propeller15, whereas in Nup214, the  interaction is not needed to  maintain the  integrity of the β-propeller  structure16. In the latter case, and probably also in Sro7, this  interaction seems to be dynamic,  suggesting that the C-terminal tail has a regulatory  function.Using the new structure as a guide, Hattendorf et al.5 have investigated how Sec9 binds Sro7 and how this interaction is related to the  function of Sro7 in yeast exocytosis. They found that part of the N-terminal domain of Sec9  associates with the convex surface of the C-terminal β- propeller of Sro7. However, the region within the Sec9 N-terminal domain involved in the interaction is poorly  conserved in other Sec9 proteins. Moreover, only  fungal Qbc-SNARE proteins contain extended N- terminal domains, and in yeast the N- terminal domain of Sec9 has been shown to be  expendable17.To explore the observed discrepancy between the binding data and the  functional  complementation assays, the authors  investigated whether the SNARE motifs of Sec9 alone are capable of interacting with Sro7. Weak binding was observed (with an  approximate affinity of 50 µM), but only when the Sro7 tail was deleted. In  addition, deletion of the tail helix moderately increased the affinity of full-length Sec9 (from 3 to 0.8 µM), suggesting that it regulates the  ability of Sro7 to interact with Sec9. This gave rise to the hypothesis that the two SNARE motifs of Sec9 compete with the tail of Sro for binding to the N-terminal β-propeller. In this case, binding may control the ability of Sec9 to enter SNARE complexes. Further s upport for this notion will require  competition  experiments between the tail and the SNARE motifs of not only Sec9 but also its Qa-SNARE partner Sso, which might prove  difficult,  considering the low  affinities.Intriguingly, the tail region of Sro is  followed by a stretch of C-terminal  heptad-repeats (Fig. 1c,d), which are not included in the new  crystal structure. This stretch was  predicted to form part of a  degenerate R-SNARE motif18, which is, in fact, less  degenerate in the Sro7  orthologs of other fungi. Tomosyn (but not the Lgl  proteins)  contains a full R-SNARE motif at the C- terminal end19,20. The  tomosyn R-SNARE motif is capable of  substituting for  synaptobrevin in forming a typical four Budding yeast is an attractive model  organism for studying cell polarity. At the onset of  asymmetric cell division, vesicles are  transported along cytoskeletal tracks to a  predefined site of the plasma membrane. Here, they undergo exocytosis, causing the  formation of a bud that enlarges to form the  daughter cell. A large  octameric protein  complex, termed the exocyst, has a key role in  directing  transport and  defining the  attachment site of the  vesicles1,2. Fusion is mediated by the SNARE proteins Snc1/2, Sso1/2 and Sec9. Two homologous  proteins, Sro7 and Sro77, bind both a  component of the exocyst (Exo84)3 and a SNARE protein (Sec9)4. Deletion of these proteins leads to a cold- sensitive defect in vesicle exocytosis,  suggesting a functional link between the  exocyst and the SNAREs. Recently, Hattendorf et al.5 have  presented the crystal structure of a large portion of Sro7, shedding the first light on the molecular mechanism by which this protein may control SNARE  function.SNAREs are evolutionary conserved  membrane proteins involved in all fusion events of the secretory pathway and are  characterized by a conserved coiled-coil  segment termed the SNARE motif. During fusion, the SNARE motifs of interacting SNAREs assemble into a parallel four-helix bundle that is initiated by the N- terminal tips and zips up toward the C-terminal membrane anchors. As a result, the membranes are pulled into close  apposition and fusion is initiated. On the basis of a highly conserved set of amino acid side chains, SNARE motifs are classified into Qa-, Qb-, Qc- and R-SNAREs, and every  functional SNARE complex contains one SNARE motif of each subfamily (reviewed in refs. 6,7). Sso1/2 (Qa), and Snc1/2 (R), each contain a single SNARE motif that is located adjacent to a C-terminal  transmembrane domain. In  contrast, Sec9 contains two SNARE motifs (Qb and Qc)  connected by a linker region and has an extended N-terminal domain.Dirk Fasshauer and Reinhard Jahn are in the Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.e-mail: dfassha@gwdg.de and rjahn@gwdg.deN E W S  A N D  V I E W S©2007 Nature Publishing Group  http://www.nature.com/nsmbNATURE STRUCTURAL & MOLECULAR BIOLOGY   VOLUME 14   NUMBER 5   MAY 2007 361helix-bundle with syntaxin-1 and SNAP-25 (Fig. 1e)18. Thus, it appears that Sro7 and Sro77 are orthologs of tomosyn, with the two yeast isoforms probably arising from a recent gene duplication. Lgl, in  contrast, is restricted to metazoans and probably has a role in cell differentiation in multicellular organisms. Apparently, an evolutionary split occurred between the two factors9,  possibly resulting from an early duplication of the tomosyn gene in animals.Considering that the R-SNARE motif of tomosyn can associate with the plasma  membrane Q-SNAREs18, the question arises whether the degenerate SNARE motif in the yeast ortholog Sro7 can bind the R-SNARE site in the yeast exocytotic SNARE complex. In fact, the complex of Sro7 and Sec9 was originally  isolated together with Sso and Snc. Hattendorf et al.5, however, did not observe a substantial change in binding affinity when the  degenerate SNARE motif was deleted, although they did not test Sec9 binding in the presence of Sso. Furthermore, in  complementation assays, a mutant Sro7 in which 42  residues were removed from the C terminus of the  degenerate SNARE motif (∆42) was still able to rescue the Sro7/Sro77  deletion strain, whereas  deletion of the entire tail region (∆142) was not  tolerated. Thus, neither of the two  structurally defined  interactions between Sro7 and the  orthologous  tomosyn and Q-SNAREs  suffices to explain the  function of these  proteins at the  molecular level. However, the new  structure provides a sound basis for future experiments, and it is likely that the association of the tail  peptide with the N- terminal propeller domain will indeed prove to be a crucial element in the function of these fascinating proteins.COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.Figure 1  Schematic overview of the Sro7 and the tomosyn SNARE complex structures. (a) Convex surface of the Sro7 structure5. (b) Side view of a. (c) Schematic of the domain structure of Sro7, colored as in a,b. The region in red between blades 10 and 11 contains phosphorylation sites in animal tomosyn and Lgl and is alternatively spliced in animal tomosyns, thus probably providing distinct binding surfaces for different regulating factors. (d) Sequence alignment of C-terminal regions of tomosyns and synaptobrevin, showing that tomosyns generally contain an R-SNARE motif, which is partially degenerated in Saccharomycotina fungi. Shown are synaptobrevin-2 from Mus musculus (MuMu_Syb2, NCBI gene ID 207626) and tomosyn homologs from Rattus norvegicus (RaNo_Tom1, 13540648), Caenorhabditis elegans (CaEl_Tom, 17508275), Drosophila melanogaster (DrMe_Tom, 28571171), Arabidopsis thaliana (ArTh_Tom1, 15239173), Magnaporthe grisea (MaGr_Tom, 39973855), Neurospora crassa (NeCr_Tom, 85090786), Aspergillus nidulans (AsNi_Tom, 40741778), Schizosaccharomyces pombe (ScPo_Tom, 19113918) and Saccharomyces cerevisiae (SaCe_Sro7p, 6325289; SaCe_Sro77, 6319362). Black highlight, synaptobrevin-2 transmembrane region; gray highlight, coiled-coil layer residues (numbers indicated at bottom). ∆42 marks position of deleted fragment5. (e) Structure of the SNARE complex of the C-terminal R-SNARE motif of tomosyn with the Q-SNAREs syntaxin-1 and SNAP-25 (ref. 18). Structure images were prepared with PyMOL (http://pymol.sourceforge.net).N E W S  A N D  V I E W S©2007 Nature Publishing Group  http://www.nature.com/nsmb362 VOLUME 14   NUMBER 5   MAY 2007   NATURE STRUCTURAL & MOLECULAR BIOLOGY1. Novick, P. et al. Biochem. Soc. Trans. 34, 683–686 (2006).2. Munson, M. & Novick, P. Nat. Struct. Mol. Biol. 13, 577–581 (2006).3. Zhang, X. et al. J. Cell Biol. 170, 273–283 (2005).4. Lehman, K., Rossi, G., Adamo, J.E. & Brennwald, P. J. Cell Biol. 146, 125–140 (1999).5. Hattendorf, D.A., Andreeva, A., Gangar, A., Brennwald, P.J. & Weis, W.I. Nature 446, 567–571 (2007).6. Hong, W. Biochim. Biophys. Acta 1744, 493–517 (2005).7. Jahn, R. & Scheller, R.H. Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).8. Humbert, P.O., Dow, L.E. & Russell, S.M. Trends Cell Biol. 16, 622-630 (2006).9. Wirtz-Peitz, F. & Knoblich, J.A. Trends Cell Biol. 16, 234–241 (2006).10. Larsson, K. et al. J. Biol. Chem. 273, 33610–33618 (1998).11. Fujita, Y. et al. Neuron 20, 905–915 (1998).12. Musch, A. et al. Mol. Biol. Cell 13, 158–168 (2002).13. Voegtli, W.C., Madrona, A.Y. & Wilson, D.K. J. Biol. Chem. 278, 34373–34379 (2003).14. Mohri, K., Vorobiev, S., Fedorov, A.A., Almo, S.C. & Ono, S. J. Biol. Chem. 279, 31697–31707 (2004).15. Appleton, B.A., Wu, P. & Wiesmann, C. Structure 14, 87–96 (2006).16. Napetschnig, J., Blobel, G. & Hoelz, A. Proc. Natl. Acad. Sci. USA 104, 1783–1788 (2007).17. Brennwald, P. et al. Cell 79, 245–258 (1994).18. Pobbati, A.V., Razeto, A., Boddener, M., Becker, S. & Fasshauer, D. J. Biol. Chem. 279, 47192–47200 (2004).19. Masuda, E.S., Huang, B.C., Fisher, J.M., Luo, Y. & Scheller, R.H. Neuron 21, 479–480 (1998).20. Fasshauer, D., Sutton, R.B., Brünger, A.T. & Jahn, R. Proc. Natl. Acad. Sci. USA 95, 15781–15786 (1998).Shifting nanoscopic clock gearsSusan S Golden, Vincent M Cassone & Andy LiWangA recent report looks at a ‘clock in a test tube’ composed of the cyanobacterial proteins KaiA, KaiB and KaiC, revealing that these dancing proteins swap partners to keep track of time.researchers and in diverse  cyanobacterial genera, have the  characteristics of circadian rhythms as described for  eukaryotic  organisms:  endogenous, self- sustained 24-hour  rhythmicity in the absence of an  externally applied cycle;  ability to reset the relative  timing of peaks to  synchronize with relevant external environmental cues; and temperature  compensation of the period4. Real progress in  understanding the  mechanism of the  cyanobacterial  circadian clock required an In 2005, the research community was  startled to learn that the cyanobacterial  circadian clock is run by an oscillator that is, ironically enough, rather mechanical. This  revelation came when Nakajima et al.1 built the  oscillator in vitro with three  proteins — KaiA, KaiB and KaiC — and ATP, and  recapitulated the  temperature- compensated circadian rhythm of  phosphorylation of the KaiC oscillator  protein. Now, several groups  coordinated by C.H. Johnson have shown that, although evolution’s  watchmaker may be blind2, the oscillator itself, once built, can be seen in four dimensions. In this study by Mori et al.3, samples from an in vitro  oscillating reaction were removed at  intervals, imaged by electron microscopy and analyzed by two- dimensional electrophoresis to determine which partners are associated in a  complex at different  circadian times. Moreover, the authors  present a  mathematical model that incorporates known behaviors of the  cyanobacterial circadian  oscillator,  accurately reproduces the rhythm of KaiC  phosphorylation and  formation of KaiABC complexes, and makes testable predictions about timekeeping states.This contribution is the latest of several  technical and conceptual leaps that have  propelled the circadian clock of the  humble  cyanobacterium Synechococcus  elongatus to fame, if not fortune, in two decades. In the first of these leaps, T.-C. Huang and  colleagues  demonstrated that daily rhythms in a  number of physiological processes, reported by  numerous Susan S. Golden, Vincent M. Cassone and Andy LiWang are in the Center for Research on Biological Clocks, Texas A&M University, College Station, Texas 77843, USA.e-mail: sgolden@tamu.edu24/01218 6KaiCKaiAATPADP ATPADPKaiBPPPPPP PPPPPPFigure 1  Schematic of the KaiABC central oscillator. Daily swings in KaiC phosphorylation are driven by rhythmic  interactions with KaiA and KaiB. Although KaiA is found associated with KaiC throughout the circadian cycle, it enhances the autokinase activity of KaiC only from circadian time 0 hours (CT0) to CT12. Around CT12, KaiC becomes  hyperphosphorylated, which induces a conformational change to a form with a higher affinity for KaiB. The KaiB-KaiC interaction results in an overall autophosphatase activity for KaiC, even in KaiB–KaiC–KaiA complexes. Once KaiC returns to its original hypophosphorylated state, at CT24, its conformation changes back to one with low affinity for KaiB, and the interaction between KaiA and KaiC starts the circadian cycle all over again. Interchange of KaiC  monomers keeps rhythmic phosphorylation of KaiC particles phase-coherent across the population, which results in a robust ensemble-averaged amplitude for several cycles. Free KaiA, KaiB and KaiC are also present throughout the cycle.Katie RisN E W S  A N D  V I E W S©2007 Nature Publishing Group  http://www.nature.com/nsmb

Budding insights on cell polarity

http://hdl.handle.net/11858/00-001M-0000-0012-E297-5

Budding insights on cell polarity

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