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could be produced that bound the Tes–LIM3 domain whereas a Mena-EVH1 domain con-taining VASP point mutations lost the ability to bind to LIM3. When the same mutations were engineered into GFP–Mena, overexpression of Tes–LIM3 was unable to displace the Mena/VASP chimaera from focal adhesions.

Competition between Tes and FPPPP ligands, demonstrated convincingly by the Way labora-tory, is not only of interest in its own right but uncovers a new mode of regulatory interactions involving LIM- and EVH1-domains. Given the 151 LIM-containing proteins in the human genome14, and multiple protein families bear-ing EVH1 domains1, there is no shortage of possibilities (Fig. 1). As far as cell migration is concerned new routes present themselves for regulating FPPPP/EVH1 interactions in differ-ent locations. The regulation of recruitment of Ena/VASP proteins to lamellipodia and filopodia tips by MRL family proteins15–17 may be used to modulate protrusion speed; similarly, regulation of interaction at focal adhesions, with zyxin and vinculin, could regulate actin-filament elonga-tion rates and lamella assembly (Fig. 2).

Finally, the potentiation of filopodia for-mation by Ena/VASP proteins6 suggests that Ena/VASP regulation influences not only

actin polymerization rates, but also the rela-tive expression of lamellipodia and filopodia. Cells cannot migrate with filopodia alone; they require lamellipodia to consolidate the advance of a protruding front. Between cell-types the relative expression of filopo-dia and lamellipodia is conspicuously vari-able (Fig. 2). Growth cones that drive axon extension are notably rich in filopodia and it is generally thought they serve a guidance function, probably through the generation of adhesions. Recent findings with fibroblasts and melanoma cells have revealed a new role for filopodia, as contributors to the construc-tion of the cytoskeleton of the lamella behind the leading front, including the initiation of stress fibre assembly18 (also M. Nemethova, S. Auinger and J. V. S., unpublished obser-vations). The backfolding of filopodia in neuronal growth cones seems, in a similar way, to contribute to the formation of the axon shaft19. It may be speculated that the differential regulation of Ena/VASP protein interactions contributes to different modes of coupling protrusion at the front and retrac-tion of the cytoskeleton behind. Does prefer-ential partnership between Mena, VASP and EVL with one of the three vertebrate WAVE

isoforms20 contribute to these phenotypic variations? Sorting out the puzzle of Ena/VASP protein functions promises interesting surprises ahead.

1. Renfranz, R. J. & Beckerle, M. C. Curr. Opin. Cell Biol. 14, 88–103 (2002).

2. Kwiatkowski, A. V., Gertler, F. B. & Loureiro, J. J. Trends Cell Biol. 13, 386–392 (2003).

3. Reinhard, M., Jarchau, T. & Ulrich, W. Trends Bioch. Sci. 26, 243–249 (2001).

4. Krause, M. et al. Ann. Rev. Cell Dev. Biol. 19, 541–564 (2003).

5. Schirenbeck, A. et al. Proc. Natl Acad. Sci. USA 103, 7694–7699 (2006).

6. Applewhite, D. A. et al. Mol. Biol. Cell 18, 2579–2591 (2007).

7. Tani, K. et al. J.Biol.Chem. 278, 21685–21692 (2003).

8. Boeda, B et al. Mol. Cell 28, 1071–1082 (2007).9. Tatarelli, C. et al. Genomics 68, 1–12 (2000).10. Tobias, E. S. et al. Oncogene 20, 2844–2853

(2001).11. Garvalov, B. K., et al. J. Cell Biol. 161, 33–39

(2003).12. Coutts, A. S. et al. J. Cell Sci. 116, 897–906

(2003).13. Prehoda, K. E., Lee, D. J. & Lim, A. Cell 97, 471–480

(1999).14. Kadrmas, J. L. & Beckerle, M. C. Nature Rev. Mol. Cell

Biol. 5, 920–921 (2004).15. Krause, M. et al. Dev. Cell 7, 571–583 (2004).16. Lafuente, E. M. et al. Dev. Cell 7, 585–595 (2004).17. Jenzora, A. et al. FEBS Lett. 579, 455–463 (2005).18. Koestler, S. A. et al. Nature Cell Biol. in the press. 19. Bray, D. & Chapman, K. J. Neurosci. 5, 3204–3213

(1985).20. Takenawa, T. & Suetsugu, S. Nature Rev. Mol. Cell Biol.

8, 37–48 (2007).

Green light for chloroplast outer-membrane proteinsJocelyn Bédard and Paul Jarvis

Most chloroplast proteins are encoded in the cell nucleus, translated in the cytosol, and targeted to the organelle in a post-translational manner. Our understanding of how proteins are targeted to the outer envelope membrane of chloroplasts has been improved with the identification of a specific soluble sorting factor.

Protein targeting to the outer envelope mem-brane of chloroplasts is still poorly understood; however, recent work focusing on a specific sub-class of chloroplast outer-membrane proteins (OMPs) — so-called signal-anchored proteins, which carry an N-terminal targeting signal — has produced a basic picture of the targeting steps involved. On page 220 of this issue, Bae et al.1 reveal a new piece in the OMP-targeting puz-zle. Specifically, they have identified an ankyrin repeat-containing protein, AKR2, as a soluble sorting factor that mediates OMP transport

from the cytosol to the organellar surface. The authors demonstrate that AKR2 possesses all the attributes necessary for it to perform this func-tion: (1) it binds specifically to OMP targeting signals; (2) it displays chaperone activity and pre-vents the aggregation of OMP cargo; (3) it binds to chloroplasts and facilitates the insertion of its cargo into the membrane.

Chloroplasts originated from an endosym-biotic event in which an early eukaryotic cell engulfed an ancestor of present-day cyanobac-teria. Similarly to their free-living relatives, they are surrounded by two distinct membranes and these are called the outer and inner envelope membranes (OM and IM, respectively). The central aqueous compartment delimited by

the envelope system is called the stroma, and contains a third membrane system — the thyla-koids — which carries the photosynthetic com-plexes. Overall, the chloroplast is believed to contain approximately 3000 different proteins2. Although this organelle possesses its own genetic system, inherited from its prokaryotic ancestor, most of its proteins are encoded in the nucleus, translated on cytosolic ribosomes, and targeted to the organelle post-translationally3.

Nucleus-encoded chloroplast proteins are typically synthesized as precursor proteins, each carrying a classical, N-terminal transit peptide . Such transit peptides direct precursor proteins to the chloroplast and mediate their translocation across the envelope membranes

Jocelyn Bédard and Paul Jarvis are at the Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK. e-mail: rpj3@le.ac.uk

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via the general import pathway3. On arrival in the stroma, the transit peptide is cleaved off to produce the mature protein. Precursor protein import through the envelope is driven by pro-tein heterocomplexes called TOC and TIC (for translocon at the outer or inner envelope mem-brane of the chloroplast). The TOC complex contains at least two receptor proteins, termed Toc34 and Toc159, and a channel-forming protein called Toc75. In contrast with standard chloroplast precursor proteins, most OMPs do not carry a classical transit peptide, and are not processed on insertion into the OM4.

Several different types of integral membrane protein have been identified in the chloroplast OM5. At present, OMPs are known to include factors involved in protein transport (TOC components), pore-forming proteins, media-tors of lipid metabolism or plastid division and proteins of unknown function. Some OMPs,

such as Toc75 and outer envelope protein, 37 kD (OEP37), are β-barrel proteins, some are poly-topic α-helical proteins (for example, OEP16), and others contain one or two α-helical trans-membrane domains (TMDs) (for example, OEP14, OEP64). The latter were identified first, and have been used most extensively in stud-ies on protein targeting to the OM. Targeting of these proteins was previously thought to be inde-pendent of proteinaceous envelope components and ATP, and was generally assumed to proceed spontaneously6. More recently, characterization of OEP14 led to the identification of an N-termi-nal sorting signal that is necessary and sufficient for targeting to the chloroplast OM7. Moreover, it was established that the insertion of OEP14 is assisted by the general protein import channel, Toc75, which seems to be able to act independ-ently of Toc34 and Toc159 receptors, and that the process requires nucleotides8–10 (Fig. 1).

Detailed analysis of the N-terminal target-ing signals of two proteins — AtOEP7 (the Arabidopsis thaliana homologue of pea OEP14) and AtOEP64 — revealed that both are very similar to those of type I signal-anchored mem-brane proteins, which are recognized by the signal recognition particle (SRP) for co-transla-tional insertion into the endoplasmic reticulum (ER) membrane11–13. In each case, the targeting sequence comprises a stretch of hydrophobic residues at the N-terminus of the protein, which also functions as an α-helical TMD. However, unlike that in ER proteins, the hydrophobic signal in chloroplast OMPs is flanked by a C-terminal positive region (CPR) that is critical for differentiating between the endomembrane system and the chloroplast OM.

Identification and characterization of these OMP targeting sequences led to the hypoth-esis that they are recognized by a soluble sort-ing factor, which assists transport to the target membrane. On page 220 of this issue, Bae et al.1 report that AKR2 is just such a targeting fac-tor. They show that two very similar isoforms of AKR2 (AKR2A and AKR2B; 81% identical), believed to have equivalent roles, are expressed in Arabidopsis. The AKR2A protein was identi-fied in a yeast two-hybrid screen for proteins that interact with AtOEP7. In vitro studies con-firmed that this interaction is specific, and indi-cated that both the N-terminal part of AKR2A and the C-terminal ankyrin repeats — degener-ate 33 amino-acid sequences that usually medi-ate protein–protein interactions — are required for binding. AKR2A interacts with the target-ing signal of AtOEP7 (including both the TMD and the CPR), as well as that of AtOEP64, but not with dysfunctional, mutated forms of these targeting signals previously shown to direct proteins to the endomembrane system, or with targeting signals for other cellular compart-ments11,12. Additionally, AKR2A bound to the C-terminal targeting signals of two Arabidopsis Toc34 isoforms (atToc33 and atToc34), imply-ing that it also mediates the transport of pro-teins with equivalent sorting information (that is, TMD plus CPR) at the C-terminus.

Bae et al.1 demonstrated that AKR2A binds to proteins carrying the OM targeting signal of AtOEP7 (at either the N-terminus or an inter-nal position) in vivo. Furthermore, these in vivo assays revealed a chaperone activity of AKR2A, preventing precursor aggregation. Such chap-erone activity is typical of soluble targeting factors, as they must maintain their substrates in an insertion-competent form. Moreover,

AKR2

7575

AR?Cytosol

OM

IM

Stroma

7

?

34159

Ribosome

Targeting signal

NTPs ?

IMS NTPs ?

Figure 1 Targeting of AtOEP7 to the chloroplast OM by AKR2. The AKR2 soluble targeting factor binds to the targeting signal (red bar) of a newly translated AtOEP7 protein in the cytosol1. The chaperone activity of AKR2 prevents the aggregation of the hydrophobic core domain of the targeting signal, and directs the protein to the chloroplast surface. At the OM, AKR2 perhaps docks at an as yet unidentified AKR2 receptor (AR?), or directly at Toc75, and facilitates the insertion of AtOEP7 into the membrane1. The insertion of signal-anchored chloroplast OM proteins is mediated by the general protein import channel, Toc75, which in this case seems to be able to function without assistance from the TOC receptors, Toc34 and Toc159 (refs 9, 10). Energy in the form of nucleotides (NTPs) is necessary for efficient insertion, but the stage or location of consumption is unclear10. Other, unidentified membrane proteins and perhaps AKR2 itself, may actively participate in the insertion process, but are not essential9. Abbreviations: IMS, intermembrane space; IM, inner envelope membrane.

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by using a combination of in vivo and in vitro experiments, the authors demonstrated that AKR2 (A and B) could improve the targeting and insertion of chloroplast OMPs. Binding affinity of AKR2 for chloroplasts is mediated by its C-terminal ankyrin repeats and can be competed, suggesting that association occurs at specific sites — that is, at a membrane-bound receptor (Fig. 1).

Finally, to investigate the role of AKR2 in planta, the authors generated plants in which the total pool of AKR2 was severely depleted1. Plants with very low levels of AKR2 protein dis-played a severe chlorotic phenotype, indicating a defect in chloroplast biogenesis; the organelles lacked a discernable thylakoid system and con-tained a large number of vesicles. The mutant plants were not only severely depleted in chlo-roplast OMPs, but also in internal, stromal and thylakoidal proteins.

The severe phenotype of the mutant Arabidopsis plants was most likely due mainly to a defect in TOC complex assembly. The atToc33 and atToc34 receptors were depleted, probably because AKR2 is required to assist their target-ing (as suggested by the fact that AKR2A binds to their C-terminal targeting signals). In turn, the Toc34 proteins are known to be involved in the targeting of Toc159 receptors, and together, both receptor components are required for the insertion of the Toc75 protein import chan-nel3. As the TOC machinery is required for the import of precursor proteins with classical transit peptides, significant depletion of TOC

complexes would be expected to have a deleteri-ous effect on organellar development.

Interestingly, AKR2A was identified in an ear-lier study by its interaction with a 14-3-3 adap-tor protein, and proposed to be important for disease resistance and oxidation metabolism14. Plants in which the expression of AKR2 was reduced, produced elevated levels of H2O2 and displayed a leaf necrosis phenotype typical of the hypersensitive response to pathogen infec-tion. The plants also showed a mild chlorotic phenotype. It is possible that the production of reactive oxygen species in these plants was an indirect consequence of a defect in chloroplast biogenesis. Nonetheless, the fact that AKR2 binds to a 14-3-3 protein raises the possibility that AKR2 also targets precursor proteins with classical transit peptides, as an unidentified 14-3-3 protein was previously proposed to be part of a precursor protein guidance complex15.

The work of Bae et al.1 raises many ques-tions. First, is AKR2 specific for chloroplast OMPs with TMD/CPR-type targeting signals, or does it also recognize other, as yet undefined signals in different types of chloroplast OMP (for example, putative internal signals in β-bar-rel proteins)? Second, what is the receptor for AKR2 at the chloroplast surface, and what other factors are involved? Third, once AKR2 docks at the chloroplast OM, how is the substrate trans-ferred to the membrane protein(s) that mediate insertion, and how does insertion occur?

Remarkably, AKR2 recognizes targeting sig-nals that are similar not only to those of type-I

membrane proteins of the ER, but even more so to those found in mitochondrial OM pro-teins16. Perhaps the N-termini of nascent chains are first screened by SRP, and then those that evade SRP-binding are recognized post-trans-lationally by distinct, soluble targeting factors for the other compartments. However, such a targeting factor has yet to be identified for mitochondrial OMPs.

Other types of chloroplast OMP have been proposed to use different targeting pathways with distinct requirements4, and so it will be interesting to determine whether these are also assisted by AKR2, or by other factors. Perhaps there is still room for 'spontaneous insertion'.

1. Bae, W. et al. Nature Cell Biol. 10, 220–227 (2008).2. Leister, D. Trends Genet. 19, 47–56 (2003).3. Bédard, J., & Jarvis, P. J. Ex. Bot. 56, 2287–2320

(2005).4. Hofmann, N. R., & Theg, S. M. Trends Plant Sci. 10,

450–457 (2005).5. Inoue, K. J. Int. Plant Biol. 49, 1100–1111 (2007).6. Schleiff, E., & Klösgen, R. B. Biochim. Biophys. Acta

1541, 22–33 (2001).7. Li, H.-M., & Chen, L.-J. Plant Cell 8, 2117–2126

(1996).8. Tu, S.-L., & Li, H.-M. Plant Cell 12, 1951–1959

(2000).9. Tu, S.-L. et al. Plant Cell 16, 2078–2088 (2004).10. Hofmann, N. R. Theg, S. M. Plant J. 44, 917–927

(2005).11. Lee, Y. J., Kim, D. H., Kim Y.-W. & Hwang, I. Plant Cell

13, 2175–2190 (2001).12. Lee, Y. J. et al. Mol. Cell 17, 281–291 (2004).13. Martoglio, B. & Dobberstein, B. Trends Cell Biol. 8,

410–415 (1998).14. Yan, J., Wang, J. & Zhang, H. Plant J. 29, 193–202

(2002).15. May, T. & Soll, J. Plant Cell 12, 53–64 (2000).16. Rapaport, D. EMBO Rep. 4, 948–952 (2003).

Elly M. Tanaka is at the Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany and the Center for Regenerative Therapies, Dresden. Gilbert Weidinger is at the Biotechnology Center of the Technical University Dresden, Tatzberg 47, D-01307 Dresden, Germany. e-mail: tanaka@mpi-cbg.de; gilbert.weidinger@biotec.tu-dresden.de

Heads or tails: can wnt tell which one is up?Elly M. Tanaka and Gilbert Weidinger

Planarian flatworms regenerate their heads and tails after amputation. it turns out that they use wnt–β-catenin signalling to determine where the head and the tail should form.

Hercules had a tough time killing the mythic Lernean hydra, a nine-headed monster whose heads kept growing back once cut. The cnidar-

ian Hydra, aptly named after that Greek beast, as well as the plathelminth flatworm Planaria, can indeed re-grow their heads; and all other parts of their bodies, for that matter. If you cut off both the head and tail from a planarian, the trunk will grow a head at the anterior wound and a tail at the posterior wound (Fig. 1a). Thus, the central body fragment retains polarity and a memory of what is missing1. Interestingly, over a century ago, T. H. Morgan observed that very

thin trunk slices will occasionally regenerate a head on both ends1. This led him to sug-gest that a gradient of material along the body determines anterior-posterior polarity and that a minimal length is required for a robust gradi-ent to form and normal polarity to be achieved. The molecular mechanisms establishing ante-rior-posterior polarity have thus far been elu-sive. Now, two new studies have shown that Wnt–β-catenin signalling determines where

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