Oiling the wheels of the endocytic pathway

296

Research Update

TRENDS in Cell Biology Vol.12 No.7 July 2002

Oiling the wheels of the endocytic pathway F. Gisou van der Goot and Jean Gruenberg An ever more complete picture of the organization and function of the endocytic pathway is emerging. New mechanisms, and in particular lipid-based mechanisms that couple membrane dynamics and sorting, are being unraveled. But the final picture is still coming into focus as new membrane domains, cell entry pathways and compartments come into view. Of special interest are the recent findings that pathogenic agents, in contrast to scientists, seem to have long discovered how to subvert membrane specialization to their own advantage.

In higher-eukaryotic cells, most receptors, as well as other cell-surface proteins and lipids, are internalized by means of clathrin-coated vesicles (CCVs) and then delivered to early endosomes. Other less-well characterized pathways, such as the caveolar route, are now drawing attention and are discussed below. After receptor–ligand uncoupling at the mildly acidic pH of the endosome (pH 6.2), housekeeping recycling receptors are segregated away from their ligands and are rapidly (t1/2 ≈ 2.5 min) transported along the recycling route and back to the cell surface, while ligands follow the degradation pathway together with downregulated receptors destined for destruction. Hence, it is generally accepted that early endosomes represent the first sorting station in the endocytic pathway. As described below, it is becoming apparent that several other mechanisms contribute to protein sorting in the degradation pathway, which consequently requires a robust sorting system. Sorting mechanisms

It has been surprisingly difficult to identify sorting signals within the cytoplasmic domain of endocytosed receptors that function in early endosomes, perhaps because multiple mechanisms operate in this pathway. While no sorting motifs have been found in recycling receptors, perhaps suggesting the existence of a default recycling pathway, sorting motifs have been found in the cytoplasmic domains of some downregulated receptors, but these bear little resemblance to each other [1]. Lysosomal targeting signals have also http://tcb.trends.com

been identified, but it is not always clear at which transport step these operate. Interestingly, transport along recycling and degradation pathways involves intermediates with characteristically different morphologies – thin tubules along the recycling pathway, and large (≈300–400 nm diameter) vesicles with numerous membrane invaginations (endosomal carrier vesicles/multivesicular bodies, ECVs/MVBs) along the degradation pathway. It has been proposed that biophysical constraints that presumably exist in the early endosomal bilayer at the neck of both forming recycling tubules and membrane invaginations act as a sorting device for lipids and proteins [1,2]. Sorting in the early endosome would thus at least partly be lipid based. Other mechanisms also contribute to protein sorting along the degradation pathway. Evidence is accumulating that implicates protein ubiquitination at several intracellular transport steps, including internalization [3,4] and degradation [5]. Recently, the tumor susceptibility gene 101 (tsg101), human VPS (hVPS)28 and the FYVE-protein HRS (see below), which are mammalian homologs of the yeast class E VPS proteins, were reported to recognize ubiquitin and to regulate the degradation of ubiquitinated proteins [48]. HRS sorts ubiquitinated membrane proteins into clathrin-coated microdomains of early endosomes [6]. These possibly correspond to the recently described bilayered clathrin coats, which are devoid of adaptor proteins and sensitive to the phosphoinositide 3-kinase inhibitor wortmannin [7]. In addition to ubiquitin itself, ubiquitination enzymes seem to play a role. The ubiquitin ligase Cbl was indeed shown to ubiquitinate the epidermal growth factor receptor (EGF-R) at the plasma membrane and to remain associated with the receptor all along the endocytic pathway, suggesting that Cbl might participate in sorting [8,9]. Recent studies now show that Cbl additionally regulates EGF-R endocytosis by recruiting CIN85 and also endophilin – an accessory protein of the endocytic machinery with acyltransferase activity [10]. Some

endocytosed proteins, in particular downregulated receptors such as EGF-R, rapidly accumulate within the membrane invaginations of multivesicular endosomes. A possible role for membrane invaginations in sorting processes is further supported by the observation that some proteins in transit that need to be reutilized also accumulate within membrane invaginations in late endosomes, including MHC class II, the mannose 6-phosphate (Man6-P) receptor and members of the tetraspanin family. These invaginations contain high amounts of an unusual phospholipid (lysobisphosphatidic acid, LBPA), which plays a role in Man6-P receptor and cholesterol transport [11,12]. Ubiquitinating enzymes might contribute to the regulation of protein movement into LBPA-rich membranes, presumably working together with other, possibly lipid-based, mechanisms [1]. Finally, phosphatidylinositol 3-phosphate (PtdIns3P) is abundant within the membrane invaginations of ECVs/MVBs [13]. The human vacuolar protein sorting protein hVPS34 appears to be required for the formation of these invaginations [14] and presumably is needed for the biogenesis of MVBs in mammalian cells [15]. Like other phosphoinositides, PtdIns3P is a low-abundance lipid that is generated by local kinases and binds with high specificity to the FYVE domain present in a relatively large family of proteins. Several FYVE-containing proteins function at the early endosomal membrane, including Rab5 GTPase effectors [16]. The yeast FYVE protein Fab1p, which is a PtdIns3P-5-OH kinase that generates phosphatidylinositol (3,5)-bisphosphate from PtdIns3P and is essential for maintenance of vacuole morphology, regulates cargo-selective sorting into the vacuole lumen [17]. Recent studies have also uncovered another phosphoinositide-binding domain – the PX or PHOX homology domain, first found in p40phox and p47phox, which are two subunits of the neutrophil oxidase involved in the production of reactive oxygen species (ROS) during defense against a range of infectious

0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(02)02307-3

Research Update

agents [18,19]. The PX domain of p40phox binds selectively to PtdIns3P, and the presence of this domain has been shown to be required for the endocytic function and/or localization of the t-SNARE Vam7 [20] and the sorting nexin SNX3 [21]. The PX domain is shared by a large number of proteins, including some involved in protein sorting and endosome function. The relationships between PtdIns3P and PX-proteins or FYVE-proteins are still unclear but should be of considerable interest in understanding the organization and sorting properties of endosomal membranes. It thus appears that several mechanisms contribute to protein sorting in the degradation pathway, which might point towards a physiological need for a robust sorting system and hence explain the difficulties in identifying individual degradation sorting signals. Moreover, it is also becoming apparent that, whatever the mechanism and molecular components involved, protein–lipid sorting, micro-heterogeneity and membrane organization all seem to be intimately coupled in endosomal membranes. Never enough: newly discovered domains, pathways and compartments

Although long mysterious, it has become increasingly clear that there are clathrinand caveolin-independent entry pathway(s) into the cell. Using dominant-negative mutants of the clathrin-interacting protein Eps15, the interleukin 2 (IL-2) receptor was shown to enter lymphocytes in a clathrin-independent manner even though these cells lacked caveolae [22]. Similarly, GPI-anchored proteins [23,24] and certain lipids such as lactosylceramide, globoside [25] and cholera-toxin-clustered GM1 [26] also enter cells independently of clathrin and caveolin-1. This clathrinindependent pathway(s) is thought to be mediated by specialized cholesterol and sphingolipid domains called lipid rafts [27], which also serve as signaling platforms at the cell surface [28]. Interestingly, although both present in plasma membrane rafts, the IL-2 receptor and GPI-anchored proteins reach different compartments upon internalization. The IL-2 receptor is subsequently routed to endosomes and lysosomes, following the classical degradation pathway, whereas GPI-anchored proteins have been shown to reach late endosomes (M. Fivaz et al., unpublished), the recycling endosome [24] or http://tcb.trends.com

TRENDS in Cell Biology Vol.12 No.7 July 2002

Caveolae

297

Rafts

Rafts

CCP

GEEC

Caveosome

?

Recycling * Early endosomes

Endoplasmic reticulum Late endosome Golgi Lysosome TRENDS in Cell Biology

Fig. 1. Schematic view of the endocytic system. In addition to the previously described organelles of the endocytic pathway (early, late and recycling endosomes), the existence of three new compartments has recently been proposed: (1) GPI-anchored-protein-enriched endosomal compartments (GEECs) [24]; (2) caveosomes that contain internalized SV40 virus while on its way to the endoplasmic reticulum (ER) along an unknown route; caveosomes receive SV40-containing caveolae from the plasma membrane and are rich in caveolin-1 (depicted in gray in cell-surface caveolar and caveosomal membranes) [32]; (3) a distinct class of endosome that might mediate clathrin-independent endocytosis from plasma membrane rafts to the Golgi and that would also contain caveolin-1 (compartment indicated by * in the figure) [26]. The relationship between this latter compartment and the caveosome is unknown at present. Well-documented transport routes are shown with green arrows, whereas recently proposed routes are shown in blue between plasma membrane rafts and endosomes [22], GEECs [24], the compartment indicated * and the Golgi complex. It is important to note that transport of rafts or raft components under normal steady-state conditions via clathrin-coated pits cannot be excluded. Transport from caveolae to the caveosomes has also been proposed [32].

the Golgi [23], possibly depending on the cell type. It therefore appears [24] that there are various clathrin- and caveolin-independent entry routes. This might be because there seem to be different types of raft at the plasma membrane, and probably also in endosomes, where sorting might well occur. Migrating cells provide the most striking recent evidence for the existence of different rafts. In motile T-lymphocytes, lipid rafts were found both at the leading edge and the uropod [29], but their lipid and protein composition differed – the ganglioside GM3 [29] and the transmembrane raft marker influenza hemagglutinin A (HA) [30] being found exclusively at the front of the cell, whereas the ganglioside GM1 occurred at the rear [29]. In addition to new entry pathways, there may be new cellular compartments. Early in the internalization into Chinese hamster ovary (CHO) cells, GPI-anchored proteins are proposed to reach, through a cdc42-dependent mechanism, a GPI-anchored-protein-enriched endosomal compartment (GEEC) that

contains internalized fluid-phase markers but neither markers of the clathrindependent pathway nor caveolin-1 [24]. GPI-anchored proteins would leave the GEEC and be transported in certain cell types to the recycling endosome and then back to the plasma membrane. This might employ a mechanism that, as for the transferrin receptor, depends both on stable, detyrosinated microtubules and on kinesin motors [31]. The GEEC appears to differ from the endocytic organelles that are traversed by molecules such as GM1 en route between the plasma membrane and the Golgi [26]. These latter endocytic organelles are supplied by a clathrinindependent mechanism, contain internalized fluid-phase markers but no markers of early and recycling endosomes and, in contrast to GEECs, contain caveolin-1 (Fig. 1). Finally, the existence of ‘caveosomes’ has been reported. This non-acidic compartment is reached by the SV40 virus at early times of infection. It contains caveolin-1 and is devoid of endocytic or

298

Research Update

Golgi markers [32] but, in contrast to the above-mentioned compartment identified by Nichols [26], is not accessible to fluidphase markers. It is not clear whether this caveolin-1-containing organelle exists in the absence of SV40 infection, or what are the constitutive functions of caveolae in endocytosis. On the one hand, caveolae have long been proposed to mediate transcytosis in endothelial cells and to have a capacity for internalization in other cells [33]. On the other, mice in which the gene encoding caveolin has been knocked out do not appear to exhibit major defects in endothelial transport [34], although defects in albumin uptake have been observed [35]. Furthermore, caveolae are highly immobile and are therefore unlikely to participate in constitutive endocytic trafficking [36]. These issues need clarification in the future: it is possible that raft and caveolar endocytic functions overlap, at least in part. Interestingly, it has recently been proposed that caveolae mediate internalization by a mechanism in which caveolin-1 acts as a negative regulator [37]. Exploiting and manipulating the endocytic pathway

Many intracellular bacterial pathogens, parasites, viruses as well as toxins use existing cellular uptake mechanisms to enter their host. Notably, lipid rafts have emerged as preferential interaction sites for a growing number of them [38,39], as most recently shown for Ebola and Marburg viruses [40], HIV [41] and Shigella flexneri (F. Lafont et al., unpublished). It has been proposed that entry by a raft-dependent mechanism prevents normal maturation of the phagosome containing the bacterium that would otherwise ultimately lead to fusion with lysosomes. However, the IL-2 receptor is internalized via rafts but transported to lysosomes – thus, rafts as such do not necessarily seem to serve as a brake for lysosomal delivery. Similarly, rafts do not prevent crosstalk with endosomes as internalized Shiga toxin B subunits seem to be transported in association with rafts to early endosomes on their way to the Golgi complex [42]. Moreover, Mycobacterium tuberculosis resides in a vacuole that shares many properties with endosomes, despite undergoing raftmediated entry [43]. The bacterium seems to interfere with the phagosome maturation process by inhibiting the recruitment of the FYVE-protein EEA1, a http://tcb.trends.com

TRENDS in Cell Biology Vol.12 No.7 July 2002

Rab5 effector, and this inhibition might be caused by the transfer of mycobacterial phosphatidyl-myo-inositol derivatives to the phagosomal membrane [44]. Bacteria have evolved numerous mechanisms to interfere with the biogenesis of phagolysosomes – a process that normally involves a complex series of interactions between phagosomes and both endosomes and lysosomes [45]. The composition of the phagosome membrane can be altered by its undesired bacterial ‘cargo’ at all stages of phagosome maturation, and it seems that each type of bacterium modifies the vacuolar membrane in its own way. New insights into this complex process are expected from the analysis of the phagosome proteome [46]. In the case of Gram-negative bacteria, alteration of the phagosomal membrane is likely to involve effector proteins of type IIIdependent secretion. An example of this was recently provided by Roy and colleagues for Legionella, which produces a protein called RalF that recruits the ADP ribosylation factor ARF to the phagosomal membrane and functions as a GDP–GTP exchange factor (GEF) for several ARF GTPases [47]. ARF proteins are involved in coat protein binding, lipid remodeling and organization of the actin cytoskeleton, and might thereby interfere with phagosome maturation at several steps. Concluding remarks

The past few years have led to an improved characterization of membrane domains and the role of lipids in trafficking, of new entry pathways and new compartments, thereby increasing the complexity of the endosomal system and highlighting the requirement for efficient sorting and targeting machineries, the existence and functioning of which should be elucidated in the future. Interesting insights should continue to come from the field of cellular microbiology. Indeed, not only are many pathways and organelles hijacked by various pathogens but some pathogens in addition are able to modify the properties of the compartment in which they reside. Future research at this interface between cell biology and infection biology is therefore expected not only to improve our understanding of the strategies used by pathogens to subvert the cell defenses of the host but also to provide new insights into the fundamental mechanisms that regulate membrane dynamics and organization.

References 1 Gruenberg, J. (2001) The endocytic pathway: a mosaic of domains. Nat. Rev. Mol. Cell Biol. 2, 721–730 2 Mukherjee, S. and Maxfield, F.R. (2000) Role of membrane organization and membrane domains in endocytic lipid trafficking. Traffic 1, 203–211 3 Hicke, L. (2001) Protein regulation by monoubiquitin. Nat. Rev. Mol. Cell Biol. 2, 195–201 4 Dupre, S. et al. (2001) Membrane transport: ubiquitylation in endosomal sorting. Curr. Biol. 11, R932–R934 5 Rocca, A. et al. (2001) Involvement of the ubiquitin/proteasome system in sorting of the interleukin 2 receptor beta chain to late endocytic compartments. Mol. Biol. Cell 12, 1293–1301 6 Raiborg, C. et al. Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat. Cell Biol. (in press) 7 Sachse, M. et al. (2002) Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting toward lysosomes. Mol. Biol. Cell 13, 1313–1328 8 de Melker, A.A. et al. (2001) c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route. J. Cell Sci. 114, 2167–2178 9 Levkowitz, G. et al. (1998) c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12, 3663–3674 10 Soubeyran, P. et al. (2002) Cbl–CIN85–endophilin complex mediates ligand-induced downregulation of EGF receptors. Nature 416, 183–187 11 Kobayashi, T. et al. (1998) A lipid associated with the antiphospholipid syndrome regulates endosome structure/function. Nature 392, 193–197 12 Kobayashi, T. et al. (1999) Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat. Cell Biol. 1, 113–118 13 Gillooly, D.J. et al. (2000) Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells EMBO J. 19, 4577–4588 14 Futter, C.E. et al. (2001) Human VPS34 is required for internal vesicle formation within multivesicular endosomes. J. Cell Biol. 155, 1251–1264 15 Fernandez-Borja, M. et al. (1999) Multivesicular body morphogenesis requires phosphatidylinositol 3-kinase activity. Curr. Biol. 9, 55–58 16 Zerial, M. and McBride, H. (2001) Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117 17 Odorizzi, G. et al. (1998) Fab1p PtdIns(3)P 5kinase function essential for protein sorting in the multivesicular body. Cell 95, 847–858 18 Kanai, F. et al. (2001) The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat. Cell Biol. 3, 675–678 19 Ellson, C.D. et al. (2001) PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40phox. Nat. Cell Biol. 3, 679–682 20 Cheever, M.L. et al. (2001) Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes. Nat. Cell Biol. 3, 613–618 21 Xu, Y. et al. (2001) SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P. Nat. Cell Biol. 3, 658–666

Research Update

22 Lamaze, C. et al. (2001) Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Mol. Cell 7, 661–671 23 Nichols, B.J. et al. (2001) Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol. 153, 529–542 24 Sabhararanjak, S. et al. (2002) GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2, 411–423 25 Puri, V. et al. (2001) Clathrin-dependent and independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J. Cell Biol. 154, 535–547 26 Nichols, B.J. A distinct class of endosome mediates clathrin-independent endocytosis to the Golgi complex. Nat. Cell Biol. (in press) 27 Nichols, B.J. and Lippincott-Schwartz, J. (2001) Endocytosis without clathrin coats. Trends Cell Biol. 11, 406–412 28 Simons, K. and Toomre, D. (2000) Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39 29 Gomez-Mouton, C. et al. (2001) Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization. Proc. Natl. Acad. Sci. U. S. A. 98, 9642–9647 30 Millan, J. et al. (2002) Lipid rafts mediate biosynthetic transport to the T lymphocyte uropod subdomain and are necessary for uropod integrity and function. Blood 99, 978–984 31 Lin, S.X. et al. (2002) Export from pericentriolar endocytic recycling compartment to cell surface depends on stable, detyrosinated (glu) microtubules and kinesin. Mol. Biol. Cell 13, 96–109 32 Pelkmans, L. et al. (2001) Caveolar endocytosis of simian virus 40 reveals a new two-step

TRENDS in Cell Biology Vol.12 No.7 July 2002

33 34

35

36

37

38

39

40

41

42

vesicular-transport pathway to the ER. Nat. Cell Biol. 3, 473–483 Parton, R.G. (2001) Cell biology. Life without caveolae. Science 293, 2404–2405 Drab, M. et al. (2001) Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293, 2449–2452 Schubert, W. et al. (2001) Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J. Biol. Chem. 276, 48619–48622 Thomsen, P. et al. (2002) Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol. Biol. Cell 13, 238–250 Le, P.U. et al. (2002) Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum. J. Biol. Chem. 277, 3371–3379 van der Goot, F.G. and Harder, T. (2001) Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack. Semin. Immunol. 13, 89–97 Norkin, L.C. et al. (2001) Association of caveolin with Chlamydia trachomatis inclusions at early and late stages of infection. Exp. Cell Res. 266, 229–238 Bavari, S. et al. (2002) Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J. Exp. Med. 195, 593–602 Nguyen, D.H. and Taub, D. (2002) CXCR4 function requires membrane cholesterol: implications for HIV infection. J. Immunol. 168, 4121–4126 Falguieres, T. et al. (2001) Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes. Mol. Biol. Cell 12, 2453–2468

299

43 Gatfield, J. and Pieters, J. (2000) Essential role for cholesterol in entry of mycobacteria into macrophages. Science 288, 1647–1650 44 Fratti, R.A. et al. (2001) Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J. Cell Biol. 154, 631–644 45 Duclos, S. and Desjardins, M. (2000) Subversion of a young phagosome: the survival strategies of intracellular pathogens. Cell. Microbiol. 2, 365–377 46 Garin, J. et al. (2001) The phagosome proteome: insight into phagosome functions. J. Cell Biol. 152, 165–180 47 Nagai, H. et al. (2002) A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295, 679–682 48 Bishop, N. et al. (2002) Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein ubiquitin conjugates. J. Cell Biol. 157, 91–101

F. Gisou van der Goot Dept of Genetics and Microbiology, CMU, 1 rue Michel Servet, University of Geneva, CH-1211 Geneva 4, Switzerland. e-mail: [email protected] Jean Gruenberg Dept of Biochemistry, 30 quai Ernest Ansermet, University of Geneva, CH-1211 Geneva 4, Switzerland.

How mitochondria import hydrophilic and hydrophobic proteins Agnieszka Chacinska, Nikolaus Pfanner and Chris Meisinger Most mitochondrial proteins are nuclear encoded and have to be transported into the organelle after synthesis on cytosolic ribosomes. Three multimeric protein complexes have been identified that import precursor proteins destined for the mitochondria: the TOM complex in the outer membrane and two TIM complexes in the inner membrane. Recent work has provided a detailed view of the different mechanisms operating during the import of the two major classes of mitochondrial proteins – hydrophilic proteins with cleavable presequences and hydrophobic proteins with multiple internal signals.

Mitochondria contain about 1000 different proteins (estimates range from 600–2000), of which only a few are synthesized in the http://tcb.trends.com

organelle directly. This implicates a requirement for a highly organized protein import system that ensures proper recognition, transport and sorting of the cytosolically synthesized proteins destined to mitochondrial subcompartments [1–3]. The signals that direct these proteins to mitochondria can be classified into two major categories (Fig. 1). The most common class contains mitochondrial precursor proteins with cleavable, N-terminal extensions, or ‘presequences’, of 20–50 amino acid residues (range between 10–80 residues), which can form amphipathic helices bearing positively charged amino acid residues on one side and a hydrophobic non-charged surface on the other side. Most of these proteins are destined for the mitochondrial matrix. The second major class of

mitochondrial precursor proteins includes many proteins of the mitochondrial inner membrane, such as the metabolite carriers. These hydrophobic precursors contain internal signal segments that are distributed throughout the entire length of the protein. Three different multimeric protein complexes localized in the two mitochondrial membranes have been identified so far (Fig. 1; Box 1). The translocase of the outer membrane (TOM complex) consists of at least seven different subunits and can be divided into a very stable core complex of 400 kDa [general import pore (GIP)] and two loosely associated receptors (Tom20, Tom70). While the presequence-carrying preproteins bind mainly to Tom20, the carrier preproteins bind preferentially to Tom70, although there can also be

0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(02)02310-3