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Sanders, S. and Field, C. M. (1995) Curr. Biol. 5, 1213–1215 Barral, Y. et al. (1999) Genes Dev. 13, 176–187 Altman, R. and Kellogg, D. R. (1997) J. Cell Biol. 138, 119–130 Tjandra, H., Compton, J. and Kellogg, D. R. (1998) Curr. Biol. 8, 991–1000 Cvrckova, F. et al. (1995) Genes Dev. 9, 1817–1830 Benton, B. K. et al. (1993) EMBO J. 12, 5267–5275 Hime, G. R., Brill, J. A. and Fuller, M. T. (1996) J. Cell Sci. 109, 2779–2788 Robinson, D. and Cooley, L. (1996) Trends Cell Biol. 6, 474–479 Novick, P., Field, C. and Schekman, R. (1980) Cell 21, 205–215 TerBush, D. R. et al. (1996) EMBO J. 15, 6483–6494 De Virgilio, C., DeMarini, D. J. and Pringle, J. R. (1996) Microbiology 142, 2897–2905 Xie, H. et al. (1999) Cell Motil. Cytoskeleton 43, 52–62 Cooper, J. A. and Kiehart, D. P. (1996) J. Cell Biol. 134, 1345–1348
Protein translocation in apoptosis Alan G. Porter In programmed cell death (apoptosis), receptor-generated or other signals are transmitted to all cellular compartments, resulting in an apoptotic cell with extensive cytoplasmic and nuclear alterations. Protein translocation is now recognized as being crucial in the induction, amplification and regulation of this process. Diverse mechanisms trigger protein translocation to and from the plasma membrane, mitochondrion and nucleus during apoptosis. This review discusseswhere, why and how the various protein-translocation events take placeand highlights their importance in the execution and regulation of apoptosis.
Programmed cell death (apoptosis) is an essential and complex regulated process for balancing cell numbers in animal development and adult homeostasis. A mammalian cell induced to die by apoptotic signals must rapidly undergo extensive and drastic structural changes, including cell and nu-
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McKie, J. M. et al. (1997) Hum. Genet. 101, 6–12 Yagi, M. et al. (1998) Gene 212, 229–236 Chant, J. and Pringle, J. (1995) J. Cell Biol. 129, 651–755 DeMarini, D. J. et al. (1997) J. Cell Biol. 139, 75–93 Flescher, E. G., Madden, K. and Snyder, M. (1993) J. Cell Biol. 122, 373–386 Giot, L. and Konopka, J. B. (1997) Mol. Biol. Cell 8, 987–998 Konopka, J. B., DeMattei, C. and Davis, C. (1995) Mol. Cell. Biol. 15, 723–730 Robinson, L. C. et al. (1999) Mol. Biol. Cell 10, 1077–1092 Bourne, H. R., Sanders, D. A. and McCormick, F. (1991) Nature 349, 117–127 Fares, H., Goetsch, L. and Pringle, J. R. (1996) J. Cell Biol. 132, 399–411 Sanders, S. and Herskowitz, I. (1996) J. Cell Biol. 134, 413–427 Kato, K. (1990) Euro. J. Neurosci. 2, 704–711
clear shrinkage, chromatin condensation, disassembly of the nuclear and cytoplasmic networks, DNA fragmentation and membrane blebbing. How does a death signal permeate to different regions of the cell and promote such drastic changes? The cytoplasmic compartment of cells contains the core apoptosis machinery in a latent or sequestered form1, and it has become increasingly apparent that its activation depends on multiple posttranslational mechanisms, especially the induced translocation of proteins. An emerging theme, discussed here, is that the permeation of the death signal to all cellular locations depends on protein translocation to and from the plasma membrane, mitochondrion and nucleus. Where, why and how protein translocation occurs in apoptosis is the focus of this review, which concentrates on some of the more recently discovered mechanisms, pathways and key players in the induction and regulation of death pathways in mammalian systems. Translocation to and from the plasma membrane The most intensively studied receptors in the tumour-necrosis factor receptor (TNFR) superfamily are TNFR1 and Fas (CD95), each of which has a ‘death domain’ in its cytoplasmic tail that is required for apoptosis signalling2. The Fas ligand– receptor system plays a role in peripheral deletion of lymphocytes and the elimination of virus-infected or cancer cells2. On the other hand, the TNF–TNFR1 system is important in the induction of inflammatory and stress responses, as well as in the apoptotic/necrotic death of cultured cells2. One breakthrough in our understanding of apoptosis signalling immediately downstream of receptors came from the discovery that the N-terminal pro-domains of the death proteases caspases 8 and 10 contain death effector domains (DEDs)2. Following ligand stimulation of receptors in the TNFR superfamily, death domain–DED interactions
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reviews mediate the specific association of caspase-8 or -10 precursors with these receptors via different classes of adaptors, including various combinations of TNFR-associated death domain (TRADD), Fasactivated death domain protein (FADD) and Fliceassociated huge protein (FLASH; Fig. 1)2,3. Stimulation of the Fas receptor induces the translocation and recruitment of FADD together with a complex of pro-caspase-8 and FLASH to the cytoplasmic tail of Fas3. Both FADD and FLASH are required for the proteolytic activation of caspase-8 that involves removal of its N-terminal DEDcontaining prodomain3. FLASH may well act as a specific chaperone and caspase activator (like the CED-4 homologue Apaf-1; see below) as it has oligomerizing activity and binds to a pro-caspase3. The proteolytically activated caspase-8, having lost its DED-containing receptor anchor2, translocates from the cytoplasmic tail to the cytoplasm, where it initiates the death cascade by proteolytic activation of downstream caspases (e.g. caspase-3) and other substrates4 (Fig. 1). Thus, translocation of DEDcontaining caspases plays a crucial role in deathreceptor signalling. As inactive pro-caspase zymogens exist in many cells, how is the receptor recruitment of pro-caspases and their inappropriate activation prevented in the absence of ligand stimulation? A protein repressor named silencer of death domains (SODD) that associates with and presumably masks the Cell-death signals (DS)
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Translocation of Bid and tBid to the mitochondrion Stunning progress in the apoptosis field has come from the realization that the mitochondrion is a source and amplifier of apoptotic signals and appears to represent an important (but not the only) conduit and commitment point to cell death6. Signals from receptors trigger the release from mitochondria6 of pro-apoptotic proteins such as apoptosis-inducing factor (AIF; see below), various caspases and, most surprisingly of all, cytochrome c (Fig. 1). Some of these receptor-derived signals induce the mitochondrial targeting of certain members of the Bcl-2 family7 (Box 1) and, consequently, the release of cytochrome c (Ref. 6). The importance of mitochondrial targeting of pro-apoptotic members of the Bcl-2 family is discussed in this and the following sections. Bid is one of at least six pro-apoptotic Bcl-2homology 3 (BH3) domain-only proteins that are members of the Bcl-2 family7 in mammals (Box 1). TNF family receptor e.g. Fas
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FIGURE 1 Protein translocation to and from the plasma membrane, mitochondrion and cytoplasm during apoptosis. Pathways (involving protein translocation) that transmit death signals are shown as a composite from different cell types. Death signals can be transmitted from the plasma membrane through the pro-apoptotic members of the Bcl-2 family7 Bid, Bax and Bad (Box 1) to mitochondria, resulting in cytochrome c release and Apaf-1–caspase-9 activation8–10,12,13,15–17,29–31. Alternatively, the mitochondrial step can be bypassed, and caspases (e.g. caspase-3) can be activated via, for example, members of the tumour-necrosis factor (TNF) receptor family and caspase-8 activation2,11. GrB is a serine protease that is delivered into target cells and activates cytoplasmic caspases in granule-mediated apoptosis52. Survival functions are coloured brown. Abbreviations: AIF, apoptosis-inducing factor50; Akt, Akt/protein kinase B18,19,21; APAF-1, apoptotic protease-activating factor-16,30,31; Bcl-XL is a member of the Bcl-2 family7 (Box 1) that can block cytochrome c release from mitochondria and/or block APAF-1-dependent caspase-9 activation7,29,34–36; Calcin, the protein phosphatase calcineurin17; CASP, caspase4,11; CYT C, cytochrome c 6,30,31; DD, death domain; FADD, Fas-activated death domain protein2; FLASH, Flice-associated huge protein3; 14–3–3, 14–3–3 protein(s)16,17,21,28; GrB, granzyme B52–54; M, mitochondrion; MEKK-1, MEK kinase-122,24,26–28; P, phosphate; PM, plasma membrane; SODD, silencer of death domains5; tBID, truncated Bid8,9.
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Alan Porter is at the Institute of Molecular and Cell Biology, An institute affiliated to The National University of Singapore, 30 Medical Drive, Singapore 117609, Republic of Singapore. E-mail: mcbagp@imcb. nus.edu.sg
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Important regulators of cell death and survival Family comprises both pro- and anti-apoptotic proteins Pro-apoptotic family members include Bid, Bax, Bad, Bak, Bik and Bim Anti-apoptotic family members include Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1 Possess Bcl-2-homology (BH) domains Can heterodimerize with each other or homodimerize See Ref. 7 for a review.
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FIGURE 2 Targeting of the pro-apoptotic Bcl-2 family members Bid, Bax and Bad to the mitochondrial membrane during apoptosis. The diagram illustrates four different known or proposed processes by which the pro-apoptotic members of the Bcl-2 family7 Bid, Bax and Bad (Box 1) translocate to and interact with mitochondria, resulting in disruption of mitochondrial functions8–10,12,13,15–17,29–31 (see also Fig. 1). In addition, cell survival is associated with the phosphorylation of Bad and its release from a mitochondrial location17–19. (a) The caspase-8 cleavage product of Bid (tBID) might bind to and activate mitochondrial Bax8,9. (b) Intact Bid might interact with mitochondrial Bax, changing it from an inactive to an active conformation10. (c) Bad is dephosphorylated by calcineurin, allowing it to interact with and neutralize the mitochondrial anti-apoptotic proteins Bcl-2 or Bcl-XL17. Conversely, phosphorylation (P) by Akt18,19 removes Bad from a mitochondrial location, and Bad becomes sequestered by 14–3–3 protein(s)16,17. (d) Cytosolic Bax translocates to the mitochondrial membrane and becomes an active homodimer12,13. Abbreviation: OM, outer mitochondrial membrane.
Bid is cleaved specifically by caspase-8, releasing a Cterminal fragment (tBID) that is targeted to mitochondria8, 9 (Figs 1 and 2a). This induces mitochondria to cluster quickly around the nucleus and release cytochrome c (Fig. 1). Cytochrome c release might depend on the heterodimerization of tBid with other Bcl-2 family members such as Bcl-XL or Bax7, which could either neutralize the antiapoptotic activity (e.g. of Bcl-XL) or activate the apoptotic activity (e.g. of Bax)8,9. The latter possibility
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is compelling because, in a different system, intact Bid translocates from the cytosol to mitochondria in a caspase-independent fashion and binds to Bax10. This, in turn, induces a conformational change in mitochondrial Bax and cytochrome c release from mitochondria10 (Fig. 2b). It appears that the caspase-8–tBID pathway provides a direct connection between the Fas receptor and mitochondria8,9, but alternative pathways do exist in those cell types where Fas-mediated apoptosis occurs independently of cytochrome c release from mitochondria2,11 (e.g. the caspase-8–caspase-3 route; Fig. 1). Translocation of Bax to the mitochondrion Bax (like tBid or Bid) can translocate to mitochondria and induce mitochondrial dysfunction and caspase activation (Fig. 1). Various apoptotic stimuli induce translocation of monomeric Bax from the cytosol to mitochondrial membranes12,13, where it inserts as a homodimer13 (Fig. 2d). It is unclear whether Bax homodimerization is a cause of its translocation or a consequence of its insertion; nevertheless, dimerization is strongly associated with cell death13. Cell-death stimuli can trigger a conformational change, exposing the N- and Ctermini of Bax, that appears to be required for its insertion into mitochondria14. However, the immediate upstream apoptotic pathway that leads to this conformational change14 and the homodimerization of Bax13 is still a mystery. Unexpectedly, Bax insertion into mitochondrial membranes causes loss of their electrical potential, release of reactive oxygen species, caspase activation and cell death without detectable cytochrome c release13. By contrast, Bax directly induces the release of cytochrome c from isolated mitochondria in a cell-free system15 and in K1- or serum-deprived cerebellar granule cells10. Bad, protein kinases and 14–3–3 proteins: emerging connections Bad is yet another pro-apoptotic protein (structurally related to Bid and Bax)7 that translocates from the cytoplasm to mitochondria during apoptosis induced in cells starved of growth factors (e.g. interleukin-3)16 or resulting from sustained increases in cytosolic free Ca21 in prostate cancer cells or neurons17. A recent study has illuminated a Ca21dependent regulatory mechanism by which Bad is induced to translocate to mitochondria and cause cell death. Following an increase in cytosolic Ca21, the Ca21-activated protein phosphatase calcineurin dephosphorylates Bad, which allows Bad to associate with mitochondria, heterodimerize with Bcl-XL and promote apoptosis17 (Figs 1 and 2c). The killing mechanism in both these examples16,17 appears to involve neutralization of the anti-apoptotic activities of mitochondrial Bcl-2 or Bcl-XL by Bad (Fig. 2c). In haemopoietic or neuronal cells treated with survival factors (e.g. interleukin-3, insulin-like growth factor 1), the receptor-associated phosphoinositide 3-kinase pathway is activated, culminating in the serine phosphorylation of Bad by the protein trends in CELL BIOLOGY (Vol. 9) October 1999
reviews kinase Akt18,19. Inhibition of calcineurin similarly results in Bad phosphorylation17. In all cases17–19, phosphorylation allows Bad to interact with a 14–3–3 protein. The resultant complex is retained in the cytosol as an inactive heterodimer, and the cell survives (Figs 1 and 2c; see also Ref. 16). Altogether, these findings show that the phosphorylation status of Bad controls its cellular localization and, consequently, cell viability in several contexts. However, the wider significance of the Akt–Bad pathway for cell survival is unclear as Bad can be phosphorylated by protein kinases other than Akt, and Bad is not expressed in all cells20. Bad is not the only substrate of Akt in the regulation of cell survival. For example, in a mechanism that is highly reminiscent of the suppression of the activity of Bad, Akt also phosphorylates the Forkhead transcription factor FKHRL1, leading to the association of FKHRL1 with a 14–3–3 protein (the zeta isoform)21. The resultant complex is retained in the cytoplasm, and the cell survives21 (Fig. 3). Withdrawal of a survival factor (insulin-like growth factor 1) from fibroblasts or cerebellar granule neurons causes the dephosphorylation of FKHRL1, which then translocates to the nucleus and activates target genes, some of which (e.g. the gene
encoding Fas ligand) are essential for mediating apoptosis21 (Fig. 3). Much evidence points to the importance of protein phosphorylation, particularly by serine/ threonine kinases, in the induction and regulation of apoptosis22. However, it has proved difficult to incorporate phosphorylation into our understanding of apoptosis pathways owing to the dearth of relevant kinase substrates and the fact that virtually all the known apoptosis-associated kinases also fulfil important functions in living cells22. During apoptosis, several protein kinases, notably MEK kinase 1 (MEKK-1), protein kinase C delta (PKCd), focaladhesion kinase and p21-activated kinase 2 (PAK2) are cleaved proteolytically and activated enzymatically by caspases4,23. Cleavage activation of MEKK-1 and PKCd can contribute to the demise of cells24,25, but how can these kinases be involved in both life and death functions23–25? Cellular localization provides a clue. In healthy cells, MEKK-1 resides exclusively in the membranous fraction of cells and activates c-Jun N-terminal kinase (JNK) by phosphorylation in a caspase-independent fashion; by contrast, the active 91-kDa caspase-derived fragment of MEKK-1 is exclusively cytosolic in genotoxin- or Fas-treated dying cells26,27. Thus, it is possible that an active Cell survival
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FIGURE 3 Protein translocation from the mitochondrion and to and from the cytoplasm and nucleus during apoptosis. Pathways (involving protein translocation) that transmit death signals are shown as a composite from different cell types. Death signals might be transmitted from the mitochondrion to the nucleus via apoptosis-inducing factor (AIF)50. At least five caspases can translocate to the nucleus during apoptosis11,23,38,42,43,45,47,48. GrB can both activate nuclear cyclin-dependent kinases (CDKs) by an unknown mechanism and cleave nuclear proteins52–54. All these various nuclear translocation events serve to promote the nuclear changes of apoptosis, including DNA degradation, chromatin condensation, specific proteolysis by caspases, transcription of death genes and inappropriate activation of CDKs. Unexpectedly, p21Cip1/Waf1 translocates from the nucleus to the cytoplasm and can either promote or prevent cell death55,56. Survival functions are coloured brown. Abbreviations/terms: Akt, Akt/protein kinase B18,19,21; ASK1, apoptosis signal-regulating kinase 1 (Ref. 56); CASP, caspase4,11; DS, death signal; 14–3–3, 14–3–3 protein(s)16,17,21,28; FasL, Fas ligand21; FKHRL1, a Forkhead transcription factor21; GrB, granzyme B52–54; ICAD/DFF-45, inhibitor of caspase-activated DNase/DNA fragmentation factor 4511,44–46; MITO, mitochondrion; PT, (mitochondrial) permeability transition6,49; P, phosphate; p21, p21Cip1/Waf1 (Ref. 55).
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reviews fragment of MEKK-1 (but not the full-length protein) translocates to the cytosol in dying cells, where it specifically phosphorylates a set of apoptotic substrates26,27. 14–3–3 proteins bind to full-length MEKK-1 but not to the 91-kDa caspase-derived fragment, suggesting that 14–3–3 proteins could be responsible for the differential subcellular locations of the two MEKK-1 species28 (Fig. 1). This is especially appealing in view of the role of 14–3–3 proteins in inhibiting the functions of phosphorylated Bad16,17 and the transcription factor FKHRL121 by altering their cellular localization (Figs 1, 2c and 3). Assembling an executioner: cytochrome c– Apaf-1–caspase-9 The translocation of cytochrome c from the intermembrane space of mitochondria to the cytoplasm is a crucial step in the transmission and amplification of many types of death signals6,29. Strong evidence from biochemical and gene-knockout approaches has showed that important cell-death pathways lie downstream of cytochrome c release29–32 (Fig. 1). Cytochrome c (together with dATP or ATP) is an essential cofactor for apoptotic protease-activating factor-1 (Apaf-1), which binds to pro-caspase-9 and induces its proteolytic activation30. It seems likely that a cytochrome-c- and nucleotide-induced conformation change in Apaf-1 leads to its oligomerization, which in turn causes the aggregation and subsequent auto-proteolytic activation of caspase-933 (Fig. 1). Two plausible mechanisms have been proposed, which are not necessarily mutually exclusive, to explain how Apaf-1/pro-caspase-9 activation is regulated. Firstly, anti-apoptotic proteins such as Bcl-2 and Bcl-XL that are tethered to the outer mitochondrial membrane could prevent cytosolic Apaf-1/procaspase-9 activation indirectly by blocking the translocation of cytochrome c from the intermembrane space29 (Fig. 1). Alternatively, Bcl-XL might inhibit caspase-9 activation directly by binding to Apaf-17 (Fig. 1). In support of this suggestion, a ternary complex of Bcl-XL, Apaf-1 and pro-caspase-9 (as well as a ternary complex comprising the equivalent Caenorhabditis elegans proteins CED-9, CED-4 and CED-3, respectively) has been observed in mammalian cells34–36, and interaction of Bcl-XL with Apaf-1 blocks Apaf-1-dependent caspase-9 activation35. So, how would the inhibition of caspase-9 activation be relieved during apoptosis? A ternary complex of Bcl-XL–Apaf-1–pro-caspase-9 could be tethered to the mitochondrial membrane via Bcl-XL. Pro-apoptotic proteins such as tBid and Bax (which induce cytochrome c release from mitochondria)8–10,15 could heterodimerize with Bcl-XL, thereby displacing Apaf1–pro-caspase-9 into the cytosol7,36. An attractive feature of this model is that it can directly link Apaf-1–pro-caspase-9 activation to the translocation of tBid, Bax or Bad to mitochondria (Fig. 1). Translocation of caspases to and from the mitochondrion Caspase-9 is capable of indirectly activating caspases 2, 6, 8 and 10, and directly activating caspase-3
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and -729,31,32 (Fig. 1). Pro-caspase-9 appears to be cytosolic in many tumour cell lines30,33. However, a substantial portion of cellular pro-caspases 2 and 9 is also found in the intermembrane space in mitochondria from various organs as well as in isolated liver mitochondria37. Following apoptosis induction or treatment with chemicals that open the mitochondrial permeability transition (PT) pore and rupture the outer mitochondrial membrane, procaspases 2 and 9 translocate from the mitochondrion to the cytosol, where they are processed to generate active enzymes37. In addition, pro-caspase9 is found almost exclusively in or associated with mitochondria of cardiomyocytes and neurons both in culture and in animals, but, in these cells, death signals induce the translocation of mitochondrial caspase-9 to the nucleus38. Compartmentalization and translocation of pro-caspase-9 (Figs 1 and 3) might be important for its regulation, especially in cells where it occupies a key upstream position in the death cascade and in organs (e.g. the mammalian brain) where it is required for proper development29,31,32. Between 10% and 90% of the caspase-3 precursor has also been detected in the intermembrane space of mitochondria from various tissues (including liver) and cell lines39,40. As with pro-caspases 2 and 9, a variety of apoptotic stimuli induce the loss of procaspase-3 from mitochondria and the appearance of active caspase-3 in the cytosol39. However, this issue remains controversial as two other studies found that pro-caspase-3 is mainly cytosolic, but undetectable in liver mitochondria32,41. Pro-caspase-7 provides the only known example of the translocation of a death protease from cytosol to mitochondria/microsomes41. Following Fasinduced apoptosis, active caspase-7 is found in liver mitochondria and microsomes, but not in the cytosol, whereas the opposite is true for active caspase341. Although caspases 3 and 7 exhibit substrate specificities indistinguishable in vitro, their in vivo substrate specificities might not be the same because their different subcellular localization might allow each of them access to a distinct set of apoptotic substrates. Nuclear translocation of caspases Several lines of evidence suggest that several procaspases or active caspases are translocated to the nucleus and trigger the specific digestion of nuclear death substrates that contributes to nuclear apoptosis (Figs 1 and 3). First, caspase-3 immunostaining is found occasionally in nuclei in some human tissues42, and caspases 1 and 3 translocate from the cytoplasm to the nuclei in regressing apoptotic neuroblastomas43. Second, several important caspase substrates perform their functions exclusively in the nucleus. These include the lamins (cleaved by caspase-6) and poly (ADP-ribose) polymerase, DNAdependent protein kinase, the retinoblastoma protein, DNA topoisomerase I and the DNA replication factor RFC-140, which are cleaved by caspase-3 or a related protease23. Caspase-3 is indispensable11 for the cleavage inactivation of nuclear inhibitor of trends in CELL BIOLOGY (Vol. 9) October 1999
reviews caspase-activated deoxyribonuclease/DNA fragmentation factor 45 (ICAD/DFF-45), generating an active CAD/DFF-40 endonuclease that is essential for the internucleosomal fragmentation (or laddering) of DNA11,44–46. A third line of evidence for functional nuclear caspases comes from the discovery of different nuclear-localization signals in the pro-domains of caspases 1 and 2 that are required for the translocation of these caspase precursors into cell nuclei47,48. Nuclear translocation of pro-caspase-1 and its activation in the nucleus only occurs after the cell is treated with a death stimulus (TNF)48. The studies discussed in this and the preceding section suggest that sequestration of pro-caspases in various cellular compartments (e.g. in the mitochondrion or cytoplasm) might restrain their activation and separate them from their substrates in living cells. Apoptosis-inducing factor: release from the mitochondrion and nuclear translocation It is clear that damage to mitochondria and the subsequent release of apoptogenic factors29 are responsible, at least in part, for the dramatic nuclear changes of apoptosis, which include chromatin condensation, disassembly of the nuclear structural network, specific proteolysis of proteins and DNA fragmentation49,50 (Figs 1 and 3). The cloning and characterization of apoptosis-inducing factor (AIF) has uncovered one crucial pathway that leads to some of these nuclear changes50. AIF, located in the intermembrane space of mitochondria in living cells50, is a 57-kDa protein homologous to bacterial ferredoxin or NADH-oxidoreductases, which is surprising as previous evidence suggested that it was a protease51. The same apoptotic stimuli that cause opening of the mitochondrial PT pore and release of cytochrome c and caspases also induce the release of AIF, which rapidly translocates to the nucleus50 (Figs 1 and 3). Recombinant AIF causes extensive nuclear chromatin condensation and massive degradation of DNA to fragments of ~50 kb when added to purified nuclei. Microinjection of AIF into the cytoplasm of living cells also causes these nuclear changes and opens the mitochondrial PT pore50. As AIF induces the release of cytochrome c and caspase9 from purified mitochondria, these results suggest that AIF might also amplify the cytochrome-c– caspase-9–caspase-3 pathway (Fig. 1). However, none of these effects of AIF is blocked by a broadspectrum caspase inhibitor50, suggesting that AIF can contribute to nuclear disintegration independently of caspase pathways that originate in mitochondria and/or the cytosol4,11,29. Granzyme B: jack-of-all-trades in granule-mediated killing Two important components of granules in natural killer and cytotoxic T cells, perforin and the serine protease granzyme B (GrB), are delivered into target cells (e.g. pathogens, tumour, non-self) by a mechanism that has been proposed to be analogous to receptor-dependent endocytosis of pathogens52. GrB is an aspartase that resembles caspases in its mode of trends in CELL BIOLOGY (Vol. 9) October 1999
action, aggressively cleaving and activating procaspases, thereby triggering the cytoplasmic death programme52 (Fig. 1). By directly activating caspases, GrB appears to evade viral mechanisms for blocking apoptosis52 and circumvent mitochondrial checkpoints such as Bcl-2 and Bcl-XL, which act upstream of most caspases7 (Fig. 1). GrB also undergoes perforin-dependent nuclear translocation in target cells52,53. Once in the nucleus, GrB directly and efficiently cleaves several proteins that are also caspase23 substrates [poly (ADP-ribose) polymerase, DNA-dependent protein kinase and nuclear mitotic apparatus protein; Fig. 3]53,54. GrB concomitantly enhances the activities of two cyclin-dependent kinases (CDKs) required for cell division (cyclinA–cdc2 and cyclin-A–CDK2), which contributes to GrB-induced killing (Fig. 3), but it is not known how they are activated and how they function in apoptosis52. The nuclear translocation of GrB, DNA fragmentation and apoptosis are all prevented by overexpression of Bcl-253. Together, these considerations emphasize the multifaceted roles of GrB and demonstrate the importance of its cytoplasmic and nuclear targeting in granule-mediated apoptosis. The cyclin-dependent kinase inhibitor p21Cip1/Waf1: a Jekyll and Hyde character The inappropriate or aberrant activation of cyclindependent kinases (cdc2 and CDKs 1, 2 and 3) is also strongly associated with apoptosis in a variety of cell-death paradigms not involving perforin/GrB (Refs 55–57 and references therein). One mechanism for the activation of nuclear cyclin–CDK2 complexes in endothelial cells undergoing apoptosis involves the specific cleavage of the CDK inhibitors p21Cip1/Waf1 and p27Kip1 by caspase-3 (or a related protease)55. A truncated p21Cip1/Waf1 lacks a C-terminal nuclear-localization signal and, consequently, exits the nucleus, thereby relieving the inhibition of CDK2 (Fig. 3). Apoptosis is partially suppressed by blocking CDK2 activation or p21Cip1/Waf1 cleavage, suggesting that activation of CDK2 as a result of the nucleus-to-cytoplasm transport of cleaved p21Cip1/Waf1 contributes to apoptosis55. It also implies that a caspase or its precursor translocates to and functions in the nucleus and raises the question of whether GrB-induced CDK2 activation52 requires a similar proteolytic cleavage mechanism. Nuclear p21Cip1/Waf1 also becomes cytoplasmic following differentiation of U937 cells into monocytes, but, in contrast to the situation in endothelial cells55, this translocation event is accompanied by resistance to various apoptotic stimuli56. Cytoplasmic p21Cip1/Waf1 forms a complex with apoptosis signal-regulating kinase 1 (ASK1) that inhibits the stress-induced mitogen-activated protein (MAP) kinase cascade in monocytes56 (Fig. 3). Overexpression of a p21Cip1/Waf1 mutant lacking the nuclear-localization signal also results in a cytoplasmic complex with ASK1 and resistance to apoptosis in U937 cells56. Monocytes destroy intracellular pathogens and extracellular non-self targets through the production of H2O2 and reactive oxygen species. Thus, the strong protective effect of cytoplasmic
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reviews p21Cip1/Waf1 might be due to inhibition of the stressinduced MAP kinase pathway22, thereby ensuring survival of monocytes in the presence of the high levels of H2O2 and reactive oxygen species that are needed to kill their targets. These results, describing the opposing functions of cytoplasmic full-length and truncated p21Cip1/Waf1 in apoptosis55,56, provide the first glimpse of the actual mechanisms (centred on translocation of p21Cip1/Waf1 to the cytoplasm) underlying the wellestablished ability of p21Cip1/Waf1 either to sensitize or to protect different cell types from apoptosis (Refs 55–57 and references therein). Conclusions and future directions It is now recognized that protein translocation plays a vital role in ensuring that the death signal is transmitted to all cellular compartments and in contributing to the drastic and extensive morphological and biochemical changes of apoptosis (summarized in Table 1). We know where many of the apoptosisrelated proteins go in the cell, but much more needs to be learnt about how the translocation processes occur and how they are regulated. Caspases are central to the overall process of apoptotic cell death4, translocating to or from the plasma membrane, mitochondrion, cytoplasm and nucleus
(Figs 1 and 3). Future studies must address the receptor specificity of the DED-containing caspases2, as well as the relative importance of the mitochondrial release of caspases and the activation of caspases that preexist in the cytosol11,32,37–41. The translocation of caspases from the cytoplasm to the nucleus has been demonstrated or inferred38,42,43,47,48, but very little is known of the substrate specificities of nuclear caspases. In general, the signals and mechanisms that determine the locations and induce the translocation of caspases are largely unknown. Three pro-apoptotic members of the Bcl-2 family7 are known to translocate to mitochondria during apoptosis and induce the release of apoptogenic proteins into the cytosol8–10,12,13,15–17. Caspase cleavage of Bid is one mechanism that induces mitochondrial localization of this class of death agonist8,9, but we need to know much more about the extra- and intracellular signals that induce the mitochondrial localization of other pro-apoptotic Bcl-2 family members (e.g. Bad and Bax; Figs 1 and 2) and how the association of these death agonists with mitochondria results in the onward transmission of the apoptotic signal12,13,16,17. Although diverse death signals induce Bid, Bax or Bad to translocate by different mechanisms, a
TABLE 1 – SUMMARY OF PROTEIN TRANSLOCATION IN APOPTOSISa Protein
Translocation from/to
Function
Refs
FADD FLASH Pro-caspase-8 Caspase-8 SODD Bid Bax Bad FKHRL1 MEKK-1 Cytochrome c Caspase-1 Caspase-2
Cytoplasm/receptor Cytoplasm/receptor Cytoplasm/receptor Receptor/cytoplasm Cytoplasm/receptor Cytoplasm/mitochondrion Cytoplasm/mitochondrion Cytoplasm/mitochondrion Cytoplasm/nucleus Membranes/cytosol Mitochondrion/cytoplasm Cytoplasm/nucleus Mitochondrion/cytoplasm. Cytoplasm/nucleus Mitochondrion/cytoplasm. Cytoplasm/nucleus Cytoplasm/nucleus Cytoplasm/mitochondrion Mitochondrion/cytoplasm. Mitochondrion/nucleus Mitochondrion/nucleus
Recruitment of caspases Recruitment of caspases Proteolytic activation Translocates to substrate(s) Inhibits receptor activation Releases death agonist(s) Releases death agonist(s) Releases death agonist(s) Induces apoptotic genes Mediates apoptosis Activates Apaf-1/caspase-9 Cleaves nuclear proteins? Cleaves cytoplasmic proteins? Cleaves nuclear proteins? Cleaves cytoplasmic proteins. Cleaves nuclear proteins Cleaves nuclear proteins ? Cleaves cytoplasmic proteins. Cleaves nuclear proteins? Promotes chromatin condensation and DNA degradation Cleaves cytoplasmic proteins. Cleaves nuclear proteins and activates CDKs Mediates apoptosis or survival
2 3 2 2 5 8,9 12,13 16,17 21 26,27 6,30,31 48 37 47 39,40 11,23,43 4,23 41 37 38
Caspase-3 Caspase-6 Caspase-7 Caspase-9 AIF Granzyme B
Outside cell/cytoplasm. Cytoplasm/nucleus
p21Cip1/Waf1
Nucleus/cytoplasm
50 52 52–54 55,56
aAbbreviations:
AIF, apoptosis-inducing factor50; Apaf-1, apoptotic protease-activating factor-16,30,31; CDKs, cyclindependent kinases52,55; FADD, Fas-activated death domain protein2; FLASH, Flice-associated huge protein3; MEKK-1, MEK kinase 122,24,26–28; SODD, silencer of death domains5.
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reviews common aspect is that, in living cells, they are sequestered in cellular compartments where they are unable to make contact with and neutralize antiapoptotic proteins or activate pro-apoptotic proteins (Fig. 2). Likewise, in the living cell, compartmentalization of caspases restrains their activation and denies them access to their substrates. 14–3–3 proteins might turn out to be important for the maintenance of cell viability by sequestering and compartmentalizing proteins such as Bad, MEKK-1 and FKHRL116,17,21,28. Thus, compartmentalization of cell-death regulators is generally important in controlling apoptosis and preventing the inappropriate activation of death agonists in living cells. AIF appears to represent a novel class of death inducer that can act independently of caspase activation50, but to what extent does it contribute to apoptosis through its ability both to release apoptogenic proteins from mitochondria and translocate to the nucleus (Figs 1 and 3)? The mechanism of action of AIF is obscure – especially as the putative NADH-oxidoreductase function is dispensable for apoptosis. Moreover, AIF does not resemble a nuclease or a protease, so how does its nuclear translocation cause chromatin condensation and degradation of DNA50? Studying the translocation of cell-death regulators will continue to contribute significantly to our understanding of apoptosis pathways. As every world traveller knows, it’s not just where you are – but where, why and how you go – that matters. References 1 Takahashi, A. and Earnshaw, W. C. (1997) Adv. Pharmacol. 41, 89–106 2 Ashkenazi, A. and Dixit, V. M. (1998) Science 281, 1305–1308 3 Imai, Y. et al. (1999) Nature 398, 777–785 4 Cryns, V. and Yuan, J. (1998) Genes Dev. 12, 1551–1570 5 Jiang, Y. et al. (1999) Science 283, 543–546 6 Green, D. R. and Reed, J. C. (1998) Science 281, 1309–1312 7 Adams, J. M. and Cory, S. (1998) Science 281, 1322–1326 8 Li, H. et al. (1998) Cell 94, 491–501 9 Luo, X. et al. (1998) Cell 94, 481–490 10 Desagher, S. et al. (1999) J. Cell Biol. 144, 891–901 11 Porter, A. G. and Jänicke, R. U. (1999) Cell Death Differ. 6, 99–104 12 Hsu, Y-T., Wolter, K. G. and Youle, R. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3668–3672 13 Gross, A. et al. (1998) EMBO J. 17, 3878–3885 14 Nechushtan, A. et al. (1999) EMBO J. 18, 2330–2341 15 Jürgensmeier, J. M. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4997–5002
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Acknowledgements I thank M. Choi and M. L. Sprengart for helpful comments on the manuscript, and the Institute of Molecular and Cell Biology, Singapore for funding.
Next month in trends in CELL BIOLOGY There will be another review on apoptosis: An emerging blueprint for apoptosis in Drosophila by John Abrams, plus: Peroxisomes: simple in function but complex in maintenance by Henk Tabak, Ineke Braakman and Ben Distel Oligosaccharide transport: pumping waste from the endoplasmic reticulum into lysosomes by Stuart Moore The world according to Arp – regulation of actin nucleation by Arp2/3 complex by Matt Welch and two articles from the translocation series: New lessons in protein targeting: how to get a folded protein across a membrane by Sarah Teter and Dan Klionsky
The mitochondrial translocation apparatus by Matthias Bauer, Sabine Hofmann, Walter Neupert and Michael Brunner trends in CELL BIOLOGY (Vol. 9) October 1999
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Protein translocation in apoptosis Alan G. Porter In programmed cell death (apoptosis), receptor-generated or other signals are transmitted to all cellular compartments, resulting in an apoptotic cell with extensive cytoplasmic and nuclear alterations. Protein translocation is now recognized as being crucial in the induction, amplification and regulation of this process. Diverse mechanisms trigger protein translocation to and from the plasma membrane, mitochondrion and nucleus during apoptosis. This review discusseswhere, why and how the various protein-translocation events take placeand highlights their importance in the execution and regulation of apoptosis.
Programmed cell death (apoptosis) is an essential and complex regulated process for balancing cell numbers in animal development and adult homeostasis. A mammalian cell induced to die by apoptotic signals must rapidly undergo extensive and drastic structural changes, including cell and nu-
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clear shrinkage, chromatin condensation, disassembly of the nuclear and cytoplasmic networks, DNA fragmentation and membrane blebbing. How does a death signal permeate to different regions of the cell and promote such drastic changes? The cytoplasmic compartment of cells contains the core apoptosis machinery in a latent or sequestered form1, and it has become increasingly apparent that its activation depends on multiple posttranslational mechanisms, especially the induced translocation of proteins. An emerging theme, discussed here, is that the permeation of the death signal to all cellular locations depends on protein translocation to and from the plasma membrane, mitochondrion and nucleus. Where, why and how protein translocation occurs in apoptosis is the focus of this review, which concentrates on some of the more recently discovered mechanisms, pathways and key players in the induction and regulation of death pathways in mammalian systems. Translocation to and from the plasma membrane The most intensively studied receptors in the tumour-necrosis factor receptor (TNFR) superfamily are TNFR1 and Fas (CD95), each of which has a ‘death domain’ in its cytoplasmic tail that is required for apoptosis signalling2. The Fas ligand– receptor system plays a role in peripheral deletion of lymphocytes and the elimination of virus-infected or cancer cells2. On the other hand, the TNF–TNFR1 system is important in the induction of inflammatory and stress responses, as well as in the apoptotic/necrotic death of cultured cells2. One breakthrough in our understanding of apoptosis signalling immediately downstream of receptors came from the discovery that the N-terminal pro-domains of the death proteases caspases 8 and 10 contain death effector domains (DEDs)2. Following ligand stimulation of receptors in the TNFR superfamily, death domain–DED interactions
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reviews mediate the specific association of caspase-8 or -10 precursors with these receptors via different classes of adaptors, including various combinations of TNFR-associated death domain (TRADD), Fasactivated death domain protein (FADD) and Fliceassociated huge protein (FLASH; Fig. 1)2,3. Stimulation of the Fas receptor induces the translocation and recruitment of FADD together with a complex of pro-caspase-8 and FLASH to the cytoplasmic tail of Fas3. Both FADD and FLASH are required for the proteolytic activation of caspase-8 that involves removal of its N-terminal DEDcontaining prodomain3. FLASH may well act as a specific chaperone and caspase activator (like the CED-4 homologue Apaf-1; see below) as it has oligomerizing activity and binds to a pro-caspase3. The proteolytically activated caspase-8, having lost its DED-containing receptor anchor2, translocates from the cytoplasmic tail to the cytoplasm, where it initiates the death cascade by proteolytic activation of downstream caspases (e.g. caspase-3) and other substrates4 (Fig. 1). Thus, translocation of DEDcontaining caspases plays a crucial role in deathreceptor signalling. As inactive pro-caspase zymogens exist in many cells, how is the receptor recruitment of pro-caspases and their inappropriate activation prevented in the absence of ligand stimulation? A protein repressor named silencer of death domains (SODD) that associates with and presumably masks the Cell-death signals (DS)
14–3–3 P Bad
?
?
Translocation of Bid and tBid to the mitochondrion Stunning progress in the apoptosis field has come from the realization that the mitochondrion is a source and amplifier of apoptotic signals and appears to represent an important (but not the only) conduit and commitment point to cell death6. Signals from receptors trigger the release from mitochondria6 of pro-apoptotic proteins such as apoptosis-inducing factor (AIF; see below), various caspases and, most surprisingly of all, cytochrome c (Fig. 1). Some of these receptor-derived signals induce the mitochondrial targeting of certain members of the Bcl-2 family7 (Box 1) and, consequently, the release of cytochrome c (Ref. 6). The importance of mitochondrial targeting of pro-apoptotic members of the Bcl-2 family is discussed in this and the following sections. Bid is one of at least six pro-apoptotic Bcl-2homology 3 (BH3) domain-only proteins that are members of the Bcl-2 family7 in mammals (Box 1). TNF family receptor e.g. Fas
SODD
Bid
GrB DD FADD
PM GrB
FLASH Bax
CASP-8
Calcin
Akt
death domain in the cytoplasmic tail of unstimulated TNFR1 appears to represent one mechanism for preventing spontaneous signalling by this receptor5. Upon TNF stimulation, SODD is released from the death domain of TNFR1, permitting the recruitment of the DED-containing adaptors needed for the binding and proteolytic activation of the apical pro-caspases, including caspases 8 and 10 (Fig. 1)5.
PRO-CASP-8
tBid Bad
M
AIF
Caspase activation
CYT C
?
CASP-3 CYT C
?
Bcl-XL APAF-1
CASP-2
CASP-7
CASP-9
14-3-3
?
MEKK-1 (Membrane-bound)
91-kDa MEKK-1 Cytosolic trends in Cell Biology
FIGURE 1 Protein translocation to and from the plasma membrane, mitochondrion and cytoplasm during apoptosis. Pathways (involving protein translocation) that transmit death signals are shown as a composite from different cell types. Death signals can be transmitted from the plasma membrane through the pro-apoptotic members of the Bcl-2 family7 Bid, Bax and Bad (Box 1) to mitochondria, resulting in cytochrome c release and Apaf-1–caspase-9 activation8–10,12,13,15–17,29–31. Alternatively, the mitochondrial step can be bypassed, and caspases (e.g. caspase-3) can be activated via, for example, members of the tumour-necrosis factor (TNF) receptor family and caspase-8 activation2,11. GrB is a serine protease that is delivered into target cells and activates cytoplasmic caspases in granule-mediated apoptosis52. Survival functions are coloured brown. Abbreviations: AIF, apoptosis-inducing factor50; Akt, Akt/protein kinase B18,19,21; APAF-1, apoptotic protease-activating factor-16,30,31; Bcl-XL is a member of the Bcl-2 family7 (Box 1) that can block cytochrome c release from mitochondria and/or block APAF-1-dependent caspase-9 activation7,29,34–36; Calcin, the protein phosphatase calcineurin17; CASP, caspase4,11; CYT C, cytochrome c 6,30,31; DD, death domain; FADD, Fas-activated death domain protein2; FLASH, Flice-associated huge protein3; 14–3–3, 14–3–3 protein(s)16,17,21,28; GrB, granzyme B52–54; M, mitochondrion; MEKK-1, MEK kinase-122,24,26–28; P, phosphate; PM, plasma membrane; SODD, silencer of death domains5; tBID, truncated Bid8,9.
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Alan Porter is at the Institute of Molecular and Cell Biology, An institute affiliated to The National University of Singapore, 30 Medical Drive, Singapore 117609, Republic of Singapore. E-mail: mcbagp@imcb. nus.edu.sg
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reviews BOX 1 – SUMMARY OF MEMBERS OF THE BCL-2 FAMILY • • • • • • •
Important regulators of cell death and survival Family comprises both pro- and anti-apoptotic proteins Pro-apoptotic family members include Bid, Bax, Bad, Bak, Bik and Bim Anti-apoptotic family members include Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1 Possess Bcl-2-homology (BH) domains Can heterodimerize with each other or homodimerize See Ref. 7 for a review.
(a)
Cytoplasm
(b)
Bid
Cytoplasm Bid
CASP-8 tBid
OM
?
OM
Bax
Bax
(c)
Cytoplasm P
OM
Bid ?
(d)
Bax
Cytoplasm
14-3-3 Bad
Bad
Bax
Bcl-XL or Bcl-2
OM Bax
Bax trends in Cell Biology
FIGURE 2 Targeting of the pro-apoptotic Bcl-2 family members Bid, Bax and Bad to the mitochondrial membrane during apoptosis. The diagram illustrates four different known or proposed processes by which the pro-apoptotic members of the Bcl-2 family7 Bid, Bax and Bad (Box 1) translocate to and interact with mitochondria, resulting in disruption of mitochondrial functions8–10,12,13,15–17,29–31 (see also Fig. 1). In addition, cell survival is associated with the phosphorylation of Bad and its release from a mitochondrial location17–19. (a) The caspase-8 cleavage product of Bid (tBID) might bind to and activate mitochondrial Bax8,9. (b) Intact Bid might interact with mitochondrial Bax, changing it from an inactive to an active conformation10. (c) Bad is dephosphorylated by calcineurin, allowing it to interact with and neutralize the mitochondrial anti-apoptotic proteins Bcl-2 or Bcl-XL17. Conversely, phosphorylation (P) by Akt18,19 removes Bad from a mitochondrial location, and Bad becomes sequestered by 14–3–3 protein(s)16,17. (d) Cytosolic Bax translocates to the mitochondrial membrane and becomes an active homodimer12,13. Abbreviation: OM, outer mitochondrial membrane.
Bid is cleaved specifically by caspase-8, releasing a Cterminal fragment (tBID) that is targeted to mitochondria8, 9 (Figs 1 and 2a). This induces mitochondria to cluster quickly around the nucleus and release cytochrome c (Fig. 1). Cytochrome c release might depend on the heterodimerization of tBid with other Bcl-2 family members such as Bcl-XL or Bax7, which could either neutralize the antiapoptotic activity (e.g. of Bcl-XL) or activate the apoptotic activity (e.g. of Bax)8,9. The latter possibility
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is compelling because, in a different system, intact Bid translocates from the cytosol to mitochondria in a caspase-independent fashion and binds to Bax10. This, in turn, induces a conformational change in mitochondrial Bax and cytochrome c release from mitochondria10 (Fig. 2b). It appears that the caspase-8–tBID pathway provides a direct connection between the Fas receptor and mitochondria8,9, but alternative pathways do exist in those cell types where Fas-mediated apoptosis occurs independently of cytochrome c release from mitochondria2,11 (e.g. the caspase-8–caspase-3 route; Fig. 1). Translocation of Bax to the mitochondrion Bax (like tBid or Bid) can translocate to mitochondria and induce mitochondrial dysfunction and caspase activation (Fig. 1). Various apoptotic stimuli induce translocation of monomeric Bax from the cytosol to mitochondrial membranes12,13, where it inserts as a homodimer13 (Fig. 2d). It is unclear whether Bax homodimerization is a cause of its translocation or a consequence of its insertion; nevertheless, dimerization is strongly associated with cell death13. Cell-death stimuli can trigger a conformational change, exposing the N- and Ctermini of Bax, that appears to be required for its insertion into mitochondria14. However, the immediate upstream apoptotic pathway that leads to this conformational change14 and the homodimerization of Bax13 is still a mystery. Unexpectedly, Bax insertion into mitochondrial membranes causes loss of their electrical potential, release of reactive oxygen species, caspase activation and cell death without detectable cytochrome c release13. By contrast, Bax directly induces the release of cytochrome c from isolated mitochondria in a cell-free system15 and in K1- or serum-deprived cerebellar granule cells10. Bad, protein kinases and 14–3–3 proteins: emerging connections Bad is yet another pro-apoptotic protein (structurally related to Bid and Bax)7 that translocates from the cytoplasm to mitochondria during apoptosis induced in cells starved of growth factors (e.g. interleukin-3)16 or resulting from sustained increases in cytosolic free Ca21 in prostate cancer cells or neurons17. A recent study has illuminated a Ca21dependent regulatory mechanism by which Bad is induced to translocate to mitochondria and cause cell death. Following an increase in cytosolic Ca21, the Ca21-activated protein phosphatase calcineurin dephosphorylates Bad, which allows Bad to associate with mitochondria, heterodimerize with Bcl-XL and promote apoptosis17 (Figs 1 and 2c). The killing mechanism in both these examples16,17 appears to involve neutralization of the anti-apoptotic activities of mitochondrial Bcl-2 or Bcl-XL by Bad (Fig. 2c). In haemopoietic or neuronal cells treated with survival factors (e.g. interleukin-3, insulin-like growth factor 1), the receptor-associated phosphoinositide 3-kinase pathway is activated, culminating in the serine phosphorylation of Bad by the protein trends in CELL BIOLOGY (Vol. 9) October 1999
reviews kinase Akt18,19. Inhibition of calcineurin similarly results in Bad phosphorylation17. In all cases17–19, phosphorylation allows Bad to interact with a 14–3–3 protein. The resultant complex is retained in the cytosol as an inactive heterodimer, and the cell survives (Figs 1 and 2c; see also Ref. 16). Altogether, these findings show that the phosphorylation status of Bad controls its cellular localization and, consequently, cell viability in several contexts. However, the wider significance of the Akt–Bad pathway for cell survival is unclear as Bad can be phosphorylated by protein kinases other than Akt, and Bad is not expressed in all cells20. Bad is not the only substrate of Akt in the regulation of cell survival. For example, in a mechanism that is highly reminiscent of the suppression of the activity of Bad, Akt also phosphorylates the Forkhead transcription factor FKHRL1, leading to the association of FKHRL1 with a 14–3–3 protein (the zeta isoform)21. The resultant complex is retained in the cytoplasm, and the cell survives21 (Fig. 3). Withdrawal of a survival factor (insulin-like growth factor 1) from fibroblasts or cerebellar granule neurons causes the dephosphorylation of FKHRL1, which then translocates to the nucleus and activates target genes, some of which (e.g. the gene
encoding Fas ligand) are essential for mediating apoptosis21 (Fig. 3). Much evidence points to the importance of protein phosphorylation, particularly by serine/ threonine kinases, in the induction and regulation of apoptosis22. However, it has proved difficult to incorporate phosphorylation into our understanding of apoptosis pathways owing to the dearth of relevant kinase substrates and the fact that virtually all the known apoptosis-associated kinases also fulfil important functions in living cells22. During apoptosis, several protein kinases, notably MEK kinase 1 (MEKK-1), protein kinase C delta (PKCd), focaladhesion kinase and p21-activated kinase 2 (PAK2) are cleaved proteolytically and activated enzymatically by caspases4,23. Cleavage activation of MEKK-1 and PKCd can contribute to the demise of cells24,25, but how can these kinases be involved in both life and death functions23–25? Cellular localization provides a clue. In healthy cells, MEKK-1 resides exclusively in the membranous fraction of cells and activates c-Jun N-terminal kinase (JNK) by phosphorylation in a caspase-independent fashion; by contrast, the active 91-kDa caspase-derived fragment of MEKK-1 is exclusively cytosolic in genotoxin- or Fas-treated dying cells26,27. Thus, it is possible that an active Cell survival
MITO
AIF DS
GrB
PT CASP-3
Caspasecleaved p21
AIF CASP-9
?
Akt
ASK1
Apoptosis
p21
CASP-6 P 14–3–3 FKHRL1
Chromatin condensation– DNA degradation to ~50-kb
Pro-CASPS 1 and 2
?
Nuclear lamins Substrates, cleaved e.g. ICAD/DFF-45
? FKHRL1
Transcription of death genes (e.g. FasL)
DNA laddering
Nuclear substrates cleaved
Cyclin A/ CDK2 or /cdc2 complex Activation
NUCLEUS
p21 CDKs
Activation blocked trends in Cell Biology
FIGURE 3 Protein translocation from the mitochondrion and to and from the cytoplasm and nucleus during apoptosis. Pathways (involving protein translocation) that transmit death signals are shown as a composite from different cell types. Death signals might be transmitted from the mitochondrion to the nucleus via apoptosis-inducing factor (AIF)50. At least five caspases can translocate to the nucleus during apoptosis11,23,38,42,43,45,47,48. GrB can both activate nuclear cyclin-dependent kinases (CDKs) by an unknown mechanism and cleave nuclear proteins52–54. All these various nuclear translocation events serve to promote the nuclear changes of apoptosis, including DNA degradation, chromatin condensation, specific proteolysis by caspases, transcription of death genes and inappropriate activation of CDKs. Unexpectedly, p21Cip1/Waf1 translocates from the nucleus to the cytoplasm and can either promote or prevent cell death55,56. Survival functions are coloured brown. Abbreviations/terms: Akt, Akt/protein kinase B18,19,21; ASK1, apoptosis signal-regulating kinase 1 (Ref. 56); CASP, caspase4,11; DS, death signal; 14–3–3, 14–3–3 protein(s)16,17,21,28; FasL, Fas ligand21; FKHRL1, a Forkhead transcription factor21; GrB, granzyme B52–54; ICAD/DFF-45, inhibitor of caspase-activated DNase/DNA fragmentation factor 4511,44–46; MITO, mitochondrion; PT, (mitochondrial) permeability transition6,49; P, phosphate; p21, p21Cip1/Waf1 (Ref. 55).
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reviews fragment of MEKK-1 (but not the full-length protein) translocates to the cytosol in dying cells, where it specifically phosphorylates a set of apoptotic substrates26,27. 14–3–3 proteins bind to full-length MEKK-1 but not to the 91-kDa caspase-derived fragment, suggesting that 14–3–3 proteins could be responsible for the differential subcellular locations of the two MEKK-1 species28 (Fig. 1). This is especially appealing in view of the role of 14–3–3 proteins in inhibiting the functions of phosphorylated Bad16,17 and the transcription factor FKHRL121 by altering their cellular localization (Figs 1, 2c and 3). Assembling an executioner: cytochrome c– Apaf-1–caspase-9 The translocation of cytochrome c from the intermembrane space of mitochondria to the cytoplasm is a crucial step in the transmission and amplification of many types of death signals6,29. Strong evidence from biochemical and gene-knockout approaches has showed that important cell-death pathways lie downstream of cytochrome c release29–32 (Fig. 1). Cytochrome c (together with dATP or ATP) is an essential cofactor for apoptotic protease-activating factor-1 (Apaf-1), which binds to pro-caspase-9 and induces its proteolytic activation30. It seems likely that a cytochrome-c- and nucleotide-induced conformation change in Apaf-1 leads to its oligomerization, which in turn causes the aggregation and subsequent auto-proteolytic activation of caspase-933 (Fig. 1). Two plausible mechanisms have been proposed, which are not necessarily mutually exclusive, to explain how Apaf-1/pro-caspase-9 activation is regulated. Firstly, anti-apoptotic proteins such as Bcl-2 and Bcl-XL that are tethered to the outer mitochondrial membrane could prevent cytosolic Apaf-1/procaspase-9 activation indirectly by blocking the translocation of cytochrome c from the intermembrane space29 (Fig. 1). Alternatively, Bcl-XL might inhibit caspase-9 activation directly by binding to Apaf-17 (Fig. 1). In support of this suggestion, a ternary complex of Bcl-XL, Apaf-1 and pro-caspase-9 (as well as a ternary complex comprising the equivalent Caenorhabditis elegans proteins CED-9, CED-4 and CED-3, respectively) has been observed in mammalian cells34–36, and interaction of Bcl-XL with Apaf-1 blocks Apaf-1-dependent caspase-9 activation35. So, how would the inhibition of caspase-9 activation be relieved during apoptosis? A ternary complex of Bcl-XL–Apaf-1–pro-caspase-9 could be tethered to the mitochondrial membrane via Bcl-XL. Pro-apoptotic proteins such as tBid and Bax (which induce cytochrome c release from mitochondria)8–10,15 could heterodimerize with Bcl-XL, thereby displacing Apaf1–pro-caspase-9 into the cytosol7,36. An attractive feature of this model is that it can directly link Apaf-1–pro-caspase-9 activation to the translocation of tBid, Bax or Bad to mitochondria (Fig. 1). Translocation of caspases to and from the mitochondrion Caspase-9 is capable of indirectly activating caspases 2, 6, 8 and 10, and directly activating caspase-3
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and -729,31,32 (Fig. 1). Pro-caspase-9 appears to be cytosolic in many tumour cell lines30,33. However, a substantial portion of cellular pro-caspases 2 and 9 is also found in the intermembrane space in mitochondria from various organs as well as in isolated liver mitochondria37. Following apoptosis induction or treatment with chemicals that open the mitochondrial permeability transition (PT) pore and rupture the outer mitochondrial membrane, procaspases 2 and 9 translocate from the mitochondrion to the cytosol, where they are processed to generate active enzymes37. In addition, pro-caspase9 is found almost exclusively in or associated with mitochondria of cardiomyocytes and neurons both in culture and in animals, but, in these cells, death signals induce the translocation of mitochondrial caspase-9 to the nucleus38. Compartmentalization and translocation of pro-caspase-9 (Figs 1 and 3) might be important for its regulation, especially in cells where it occupies a key upstream position in the death cascade and in organs (e.g. the mammalian brain) where it is required for proper development29,31,32. Between 10% and 90% of the caspase-3 precursor has also been detected in the intermembrane space of mitochondria from various tissues (including liver) and cell lines39,40. As with pro-caspases 2 and 9, a variety of apoptotic stimuli induce the loss of procaspase-3 from mitochondria and the appearance of active caspase-3 in the cytosol39. However, this issue remains controversial as two other studies found that pro-caspase-3 is mainly cytosolic, but undetectable in liver mitochondria32,41. Pro-caspase-7 provides the only known example of the translocation of a death protease from cytosol to mitochondria/microsomes41. Following Fasinduced apoptosis, active caspase-7 is found in liver mitochondria and microsomes, but not in the cytosol, whereas the opposite is true for active caspase341. Although caspases 3 and 7 exhibit substrate specificities indistinguishable in vitro, their in vivo substrate specificities might not be the same because their different subcellular localization might allow each of them access to a distinct set of apoptotic substrates. Nuclear translocation of caspases Several lines of evidence suggest that several procaspases or active caspases are translocated to the nucleus and trigger the specific digestion of nuclear death substrates that contributes to nuclear apoptosis (Figs 1 and 3). First, caspase-3 immunostaining is found occasionally in nuclei in some human tissues42, and caspases 1 and 3 translocate from the cytoplasm to the nuclei in regressing apoptotic neuroblastomas43. Second, several important caspase substrates perform their functions exclusively in the nucleus. These include the lamins (cleaved by caspase-6) and poly (ADP-ribose) polymerase, DNAdependent protein kinase, the retinoblastoma protein, DNA topoisomerase I and the DNA replication factor RFC-140, which are cleaved by caspase-3 or a related protease23. Caspase-3 is indispensable11 for the cleavage inactivation of nuclear inhibitor of trends in CELL BIOLOGY (Vol. 9) October 1999
reviews caspase-activated deoxyribonuclease/DNA fragmentation factor 45 (ICAD/DFF-45), generating an active CAD/DFF-40 endonuclease that is essential for the internucleosomal fragmentation (or laddering) of DNA11,44–46. A third line of evidence for functional nuclear caspases comes from the discovery of different nuclear-localization signals in the pro-domains of caspases 1 and 2 that are required for the translocation of these caspase precursors into cell nuclei47,48. Nuclear translocation of pro-caspase-1 and its activation in the nucleus only occurs after the cell is treated with a death stimulus (TNF)48. The studies discussed in this and the preceding section suggest that sequestration of pro-caspases in various cellular compartments (e.g. in the mitochondrion or cytoplasm) might restrain their activation and separate them from their substrates in living cells. Apoptosis-inducing factor: release from the mitochondrion and nuclear translocation It is clear that damage to mitochondria and the subsequent release of apoptogenic factors29 are responsible, at least in part, for the dramatic nuclear changes of apoptosis, which include chromatin condensation, disassembly of the nuclear structural network, specific proteolysis of proteins and DNA fragmentation49,50 (Figs 1 and 3). The cloning and characterization of apoptosis-inducing factor (AIF) has uncovered one crucial pathway that leads to some of these nuclear changes50. AIF, located in the intermembrane space of mitochondria in living cells50, is a 57-kDa protein homologous to bacterial ferredoxin or NADH-oxidoreductases, which is surprising as previous evidence suggested that it was a protease51. The same apoptotic stimuli that cause opening of the mitochondrial PT pore and release of cytochrome c and caspases also induce the release of AIF, which rapidly translocates to the nucleus50 (Figs 1 and 3). Recombinant AIF causes extensive nuclear chromatin condensation and massive degradation of DNA to fragments of ~50 kb when added to purified nuclei. Microinjection of AIF into the cytoplasm of living cells also causes these nuclear changes and opens the mitochondrial PT pore50. As AIF induces the release of cytochrome c and caspase9 from purified mitochondria, these results suggest that AIF might also amplify the cytochrome-c– caspase-9–caspase-3 pathway (Fig. 1). However, none of these effects of AIF is blocked by a broadspectrum caspase inhibitor50, suggesting that AIF can contribute to nuclear disintegration independently of caspase pathways that originate in mitochondria and/or the cytosol4,11,29. Granzyme B: jack-of-all-trades in granule-mediated killing Two important components of granules in natural killer and cytotoxic T cells, perforin and the serine protease granzyme B (GrB), are delivered into target cells (e.g. pathogens, tumour, non-self) by a mechanism that has been proposed to be analogous to receptor-dependent endocytosis of pathogens52. GrB is an aspartase that resembles caspases in its mode of trends in CELL BIOLOGY (Vol. 9) October 1999
action, aggressively cleaving and activating procaspases, thereby triggering the cytoplasmic death programme52 (Fig. 1). By directly activating caspases, GrB appears to evade viral mechanisms for blocking apoptosis52 and circumvent mitochondrial checkpoints such as Bcl-2 and Bcl-XL, which act upstream of most caspases7 (Fig. 1). GrB also undergoes perforin-dependent nuclear translocation in target cells52,53. Once in the nucleus, GrB directly and efficiently cleaves several proteins that are also caspase23 substrates [poly (ADP-ribose) polymerase, DNA-dependent protein kinase and nuclear mitotic apparatus protein; Fig. 3]53,54. GrB concomitantly enhances the activities of two cyclin-dependent kinases (CDKs) required for cell division (cyclinA–cdc2 and cyclin-A–CDK2), which contributes to GrB-induced killing (Fig. 3), but it is not known how they are activated and how they function in apoptosis52. The nuclear translocation of GrB, DNA fragmentation and apoptosis are all prevented by overexpression of Bcl-253. Together, these considerations emphasize the multifaceted roles of GrB and demonstrate the importance of its cytoplasmic and nuclear targeting in granule-mediated apoptosis. The cyclin-dependent kinase inhibitor p21Cip1/Waf1: a Jekyll and Hyde character The inappropriate or aberrant activation of cyclindependent kinases (cdc2 and CDKs 1, 2 and 3) is also strongly associated with apoptosis in a variety of cell-death paradigms not involving perforin/GrB (Refs 55–57 and references therein). One mechanism for the activation of nuclear cyclin–CDK2 complexes in endothelial cells undergoing apoptosis involves the specific cleavage of the CDK inhibitors p21Cip1/Waf1 and p27Kip1 by caspase-3 (or a related protease)55. A truncated p21Cip1/Waf1 lacks a C-terminal nuclear-localization signal and, consequently, exits the nucleus, thereby relieving the inhibition of CDK2 (Fig. 3). Apoptosis is partially suppressed by blocking CDK2 activation or p21Cip1/Waf1 cleavage, suggesting that activation of CDK2 as a result of the nucleus-to-cytoplasm transport of cleaved p21Cip1/Waf1 contributes to apoptosis55. It also implies that a caspase or its precursor translocates to and functions in the nucleus and raises the question of whether GrB-induced CDK2 activation52 requires a similar proteolytic cleavage mechanism. Nuclear p21Cip1/Waf1 also becomes cytoplasmic following differentiation of U937 cells into monocytes, but, in contrast to the situation in endothelial cells55, this translocation event is accompanied by resistance to various apoptotic stimuli56. Cytoplasmic p21Cip1/Waf1 forms a complex with apoptosis signal-regulating kinase 1 (ASK1) that inhibits the stress-induced mitogen-activated protein (MAP) kinase cascade in monocytes56 (Fig. 3). Overexpression of a p21Cip1/Waf1 mutant lacking the nuclear-localization signal also results in a cytoplasmic complex with ASK1 and resistance to apoptosis in U937 cells56. Monocytes destroy intracellular pathogens and extracellular non-self targets through the production of H2O2 and reactive oxygen species. Thus, the strong protective effect of cytoplasmic
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reviews p21Cip1/Waf1 might be due to inhibition of the stressinduced MAP kinase pathway22, thereby ensuring survival of monocytes in the presence of the high levels of H2O2 and reactive oxygen species that are needed to kill their targets. These results, describing the opposing functions of cytoplasmic full-length and truncated p21Cip1/Waf1 in apoptosis55,56, provide the first glimpse of the actual mechanisms (centred on translocation of p21Cip1/Waf1 to the cytoplasm) underlying the wellestablished ability of p21Cip1/Waf1 either to sensitize or to protect different cell types from apoptosis (Refs 55–57 and references therein). Conclusions and future directions It is now recognized that protein translocation plays a vital role in ensuring that the death signal is transmitted to all cellular compartments and in contributing to the drastic and extensive morphological and biochemical changes of apoptosis (summarized in Table 1). We know where many of the apoptosisrelated proteins go in the cell, but much more needs to be learnt about how the translocation processes occur and how they are regulated. Caspases are central to the overall process of apoptotic cell death4, translocating to or from the plasma membrane, mitochondrion, cytoplasm and nucleus
(Figs 1 and 3). Future studies must address the receptor specificity of the DED-containing caspases2, as well as the relative importance of the mitochondrial release of caspases and the activation of caspases that preexist in the cytosol11,32,37–41. The translocation of caspases from the cytoplasm to the nucleus has been demonstrated or inferred38,42,43,47,48, but very little is known of the substrate specificities of nuclear caspases. In general, the signals and mechanisms that determine the locations and induce the translocation of caspases are largely unknown. Three pro-apoptotic members of the Bcl-2 family7 are known to translocate to mitochondria during apoptosis and induce the release of apoptogenic proteins into the cytosol8–10,12,13,15–17. Caspase cleavage of Bid is one mechanism that induces mitochondrial localization of this class of death agonist8,9, but we need to know much more about the extra- and intracellular signals that induce the mitochondrial localization of other pro-apoptotic Bcl-2 family members (e.g. Bad and Bax; Figs 1 and 2) and how the association of these death agonists with mitochondria results in the onward transmission of the apoptotic signal12,13,16,17. Although diverse death signals induce Bid, Bax or Bad to translocate by different mechanisms, a
TABLE 1 – SUMMARY OF PROTEIN TRANSLOCATION IN APOPTOSISa Protein
Translocation from/to
Function
Refs
FADD FLASH Pro-caspase-8 Caspase-8 SODD Bid Bax Bad FKHRL1 MEKK-1 Cytochrome c Caspase-1 Caspase-2
Cytoplasm/receptor Cytoplasm/receptor Cytoplasm/receptor Receptor/cytoplasm Cytoplasm/receptor Cytoplasm/mitochondrion Cytoplasm/mitochondrion Cytoplasm/mitochondrion Cytoplasm/nucleus Membranes/cytosol Mitochondrion/cytoplasm Cytoplasm/nucleus Mitochondrion/cytoplasm. Cytoplasm/nucleus Mitochondrion/cytoplasm. Cytoplasm/nucleus Cytoplasm/nucleus Cytoplasm/mitochondrion Mitochondrion/cytoplasm. Mitochondrion/nucleus Mitochondrion/nucleus
Recruitment of caspases Recruitment of caspases Proteolytic activation Translocates to substrate(s) Inhibits receptor activation Releases death agonist(s) Releases death agonist(s) Releases death agonist(s) Induces apoptotic genes Mediates apoptosis Activates Apaf-1/caspase-9 Cleaves nuclear proteins? Cleaves cytoplasmic proteins? Cleaves nuclear proteins? Cleaves cytoplasmic proteins. Cleaves nuclear proteins Cleaves nuclear proteins ? Cleaves cytoplasmic proteins. Cleaves nuclear proteins? Promotes chromatin condensation and DNA degradation Cleaves cytoplasmic proteins. Cleaves nuclear proteins and activates CDKs Mediates apoptosis or survival
2 3 2 2 5 8,9 12,13 16,17 21 26,27 6,30,31 48 37 47 39,40 11,23,43 4,23 41 37 38
Caspase-3 Caspase-6 Caspase-7 Caspase-9 AIF Granzyme B
Outside cell/cytoplasm. Cytoplasm/nucleus
p21Cip1/Waf1
Nucleus/cytoplasm
50 52 52–54 55,56
aAbbreviations:
AIF, apoptosis-inducing factor50; Apaf-1, apoptotic protease-activating factor-16,30,31; CDKs, cyclindependent kinases52,55; FADD, Fas-activated death domain protein2; FLASH, Flice-associated huge protein3; MEKK-1, MEK kinase 122,24,26–28; SODD, silencer of death domains5.
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reviews common aspect is that, in living cells, they are sequestered in cellular compartments where they are unable to make contact with and neutralize antiapoptotic proteins or activate pro-apoptotic proteins (Fig. 2). Likewise, in the living cell, compartmentalization of caspases restrains their activation and denies them access to their substrates. 14–3–3 proteins might turn out to be important for the maintenance of cell viability by sequestering and compartmentalizing proteins such as Bad, MEKK-1 and FKHRL116,17,21,28. Thus, compartmentalization of cell-death regulators is generally important in controlling apoptosis and preventing the inappropriate activation of death agonists in living cells. AIF appears to represent a novel class of death inducer that can act independently of caspase activation50, but to what extent does it contribute to apoptosis through its ability both to release apoptogenic proteins from mitochondria and translocate to the nucleus (Figs 1 and 3)? The mechanism of action of AIF is obscure – especially as the putative NADH-oxidoreductase function is dispensable for apoptosis. Moreover, AIF does not resemble a nuclease or a protease, so how does its nuclear translocation cause chromatin condensation and degradation of DNA50? Studying the translocation of cell-death regulators will continue to contribute significantly to our understanding of apoptosis pathways. As every world traveller knows, it’s not just where you are – but where, why and how you go – that matters. References 1 Takahashi, A. and Earnshaw, W. C. (1997) Adv. Pharmacol. 41, 89–106 2 Ashkenazi, A. and Dixit, V. M. (1998) Science 281, 1305–1308 3 Imai, Y. et al. (1999) Nature 398, 777–785 4 Cryns, V. and Yuan, J. (1998) Genes Dev. 12, 1551–1570 5 Jiang, Y. et al. (1999) Science 283, 543–546 6 Green, D. R. and Reed, J. C. (1998) Science 281, 1309–1312 7 Adams, J. M. and Cory, S. (1998) Science 281, 1322–1326 8 Li, H. et al. (1998) Cell 94, 491–501 9 Luo, X. et al. (1998) Cell 94, 481–490 10 Desagher, S. et al. (1999) J. Cell Biol. 144, 891–901 11 Porter, A. G. and Jänicke, R. U. (1999) Cell Death Differ. 6, 99–104 12 Hsu, Y-T., Wolter, K. G. and Youle, R. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3668–3672 13 Gross, A. et al. (1998) EMBO J. 17, 3878–3885 14 Nechushtan, A. et al. (1999) EMBO J. 18, 2330–2341 15 Jürgensmeier, J. M. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4997–5002
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Acknowledgements I thank M. Choi and M. L. Sprengart for helpful comments on the manuscript, and the Institute of Molecular and Cell Biology, Singapore for funding.
Next month in trends in CELL BIOLOGY There will be another review on apoptosis: An emerging blueprint for apoptosis in Drosophila by John Abrams, plus: Peroxisomes: simple in function but complex in maintenance by Henk Tabak, Ineke Braakman and Ben Distel Oligosaccharide transport: pumping waste from the endoplasmic reticulum into lysosomes by Stuart Moore The world according to Arp – regulation of actin nucleation by Arp2/3 complex by Matt Welch and two articles from the translocation series: New lessons in protein targeting: how to get a folded protein across a membrane by Sarah Teter and Dan Klionsky
The mitochondrial translocation apparatus by Matthias Bauer, Sabine Hofmann, Walter Neupert and Michael Brunner trends in CELL BIOLOGY (Vol. 9) October 1999
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