Vesicular Protein Transport

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Vesicular protein transport

P Schu ABSTRACT Protein transport and sorting in the secretory and endocytic pathways via Georg-August-Universita¨tGo¨ttingen, Zentrum vesicles is required for organelle biogenesis, constitutive and regulated Biochemie und Molekulare Zellbiologie, secretion and constitutive and regulated endocytosis. It is essential for a Go¨ttingen, Germany multicellular organism and the function of its specialised cell types that the Correspondence: multiple transport and sorting events are highly accurate. They determine the P Schu, Georg-August-Universita¨t protein and lipid composition of specialised compartments, receptor protein Go¨ttingen, Zentrum Biochemie und function and membrane homeostasis. This review describes the individual Molekulare Zellbiologie, Biochemie II, events involved in the process of vesicle mediated protein transport and sort- Heinrich-Du¨ker-Weg 12, D-37073, Go¨ttingen, Germany ing and summarizes the knowledge about the function of proteins and lipids Tel: ϩ49 551 395949 orchestrating the process. The Pharmacogenomics Journal (2001) 1, 262–271. Fax: ϩ49 551 395979 E-mail: pschuȰgwdg.de Keywords: adaptor; clathrin; phosphatidylinositol; rab; vesicular transport

Vesicular protein transport and sorting between the intracellular compartments of the biosynthetic and secretory pathway and the compartments of the endocytic route starting at the plasma membrane requires the formation of coated- vesicles. The proteins forming the coat bind to the cargo proteins of the vesicles so that transport vesicle formation and protein sorting appear to be linked pro- cesses. The proteins and mechanisms mediating vesicular protein transport are conserved from yeast to man. As we learn more about the mechanisms and proteins regulating vesicular protein transport we realize how many diseases are caused by defects in vesicular protein transport. This review summarizes aspects and mechanisms required for vesicular protein transport. Those interested in reading more about particular aspects will ﬁnd extensive reviews in the literature. Protein sorting events at the trans-Golgi network include protein transport to endosomes and lysosomes, to plasma membrane domains eg basolateral or apical, and the formation of various secretory organelles and constitutive secretion making regulation of protein sorting at this organelle especially complex. Endocytosis at the plasma membrane is a comparably simple event, but endo- cytosed proteins can recycle back to the cell surface and in polarized cells they can be transported to a different plasma membrane domain. They can also be stored in vesicles before they recycle back to the cell surface. Others are transported back to the trans-Golgi network or they are transported to lysosomes for degradation. There are two coat protein complexes, COP-I and COP-II, which mediate transport between the endoplasmic reticulum and the Golgi as well as within the Golgi.1 However there are 10 coat protein complexes and proteins known, which are involved in protein sorting between the trans-Golgi network and the plasma membrane. The family of heterotetrameric adaptor protein complexes mediating vesicular protein transport in post-Golgi compartments and the ER/Golgi COP 2 Received: 3 May 2001 complexes are phylogenetically related. They are all ubiquitously expressed in Revised: 29 August 2001 mammals and many of these also exist as tissue-speciﬁc isoforms encoded by Accepted: 4 September 2001 different genes or generated by alternative splicing. Formation of transport ves- Vesicular protein transport P Schu 263

Figure 1 post-Golgi protein trafficking pathways and the vesicle coat proteins and heterotetrameric coat protein complexes mediating protein sorting. icles and protein sorting by coat proteins is compartment specific. The AP-2 complex for example mediates vesicle formation and protein sorting only at the plasma membrane. Accessory proteins regulating vesicle budding and formation, vesicle transport and vesicle fusion with the proper target membrane bind to specialized domains of the coat proteins. They also interact with each other generating a complex regulatory network we are just beginning to understand. Lipid metabolism is required for proper protein sorting and phosphatidylinositol phosphorylation and dephosphorylation is a key regulatory element. Coat proteins and accessory proteins specifically bind to these phosphoinositides. Finally there is a family of proteins ensuring fusion of the vesicle with the proper target membrane. Some of these are membrane proteins and are part of the protein coat, whereas others cycle between the membrane and the cytoplasm regulated by protein phosphorylation and GTPase cycles. Figure 2 Composition of heterotetrameric adaptor-protein complexes as described in Table 1. Arrows indicate subunit interactions. ?: Interaction of ␮- and ␴-adaptins is indicated by ␮1-deficient cells, HETEROTETRAMERIC ADAPTOR-PROTEIN which express reduced levels of ␴1 (see Heterotetrameric Adaptor- COMPLEXES Protein Complexes). A family of four heterotetrameric coat-protein complexes (Figures 1 and 2), AP-1, AP-2, AP-3 and AP-4 exists. All consist of two large subunits of ෂ100 kDa. Both large subunits of the complexes consist of the N-terminal globular domain in AP-2, ␦ in AP-3 and ⑀ in AP-4. The second large subunit and the C-terminal globular domain, the so called ‘ear’ of the complexes shows the highest sequence conservation domain. They are separated by a proline-rich flexible stalk. among the AP-subunits, which are all also called adaptins. One subunit of these pairs shows limited sequence hom- These are named ␤1- to ␤4-adaptin. The AP-complexes ology among the AP-complexes and is named ␥ in AP-1, ␣ further contain one medium sized ␮-adaptin of ෂ50 kDa and

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Table 1 Subunit composition of the heterotetrameric AP-3 and AP-4 could bind to the trans-Golgi network as well 3–11 adaptor-protein complexes as to endosomes. Immunolocalization as well as protein sorting analysis in AP-1-deficient cell lines indicate that AP- Complex Large Medium Small Large 1 may also be involved in endosomal protein sorting.12,13 (variable) Natural mouse mutants deficient in adaptins of the AP-3 complex are viable and are a model for the Hermansky-Pud- ␥ ␮ ␴ ␴ ␤ AP-1A 1 1A 1A/ 1B 1 lack-Syndrome described in humans. Patients have defects ␥ ␮ ␴ ␴ ␤ AP-1B 1 1B 1A/ 1B 1 in the biosynthesis of specialized lysosomes and show pig- vAP-1A ␥2?␴1A/␴1B ? mentation defects, impaired blood coagulation and ceroid AP-2 ␣A (large) ␮2 ␴2 ␤2 deposition.14–16 Mouse ‘knock-out’s of AP-1 adaptin sub- ␣A (small) units are however embryonic lethal.13,17 ␣ C The AP-1 complex exists as the ubiquitous AP-1A and the AP-3A ␦␮3A ␴3A ␤3A polarized epithelia specific AP-1B complex. The AP-1B com- AP-3B ␦␮3B ␴3B ␤3B plex contains the ␮1B-adaptin, which is 87% homologous to the ubiquitously ␮1A-adaptin.18 Other epithelia-specific AP-4 ⑀␮4 ␴4 ␤4 AP-1 adaptin isoforms are not known. An AP-1 related adap- AP-1A and the variant vAP-1A are ubiquitously expressed, AP-1B is only expressed tor complex exists at the late Golgi, but it does not colocalise in polarized epithelial cells. AP-2 complexes are ubiquitously expressed: neuron with clathrin. It contains the ␥2-adaptin homologous to the specific ␣A (large) is 22 amino acids longer than the ubiquitously expressed ␣A. AP-1 ␥-adaptin. ␴1 adaptin is present in two ubiquitously AP-3A is ubiquitously expressed, AP-3B is only expressed in neuronal cells. AP-4 ␴ ␴ ␴ ␴ is ubiquitously expressed. expressed isoforms, 1A and 1B. Both 1A and 1B interact in vitro with ␥-adaptin and with the homologous ␥2-adaptin. Other subunits of such a putative heterotetrameric like vAP- ␴ ෂ one small -adaptin subunit of 20 kDa (Table 1). AP-1 and 1 (variant) complex are not known.19,20 AP-2 are localised to the trans-Golgi network and to the The AP-2 complex consists in two isoforms containing dif- plasma membrane respectively and both mediate clathrin ␣ ␣ ␣ ferent -adaptins, a 108-kDa A-adaptin and a 104-kDa C- binding and formation of a clathrin-basket as the outer layer adaptin. Both isoforms are encoded by different genes and of the coat (Figures 1 and 3, see Clathrin). AP-4 does not are ubiquitously expressed. Their relative expression levels bind clathrin, whereas AP-3 may or may not bind clathrin. however vary, indicating that they fullfill different functions, although both isoforms are found on the same ves- ␣ icles. A-adaptin RNA is alternatively spliced in the brain, introducing an additional sequence into the proline-rich flexible stack which links the N- and C-terminal globular domains.21 The AP-3 complex is present as the ubiquitously expressed AP-3A and the neuron-specific AP-3B complex. Both share the ␦-adaptin, but AP-3B contains the ␤3B, ␮3B and ␴3B adaptins.5,6,22 The AP-4 complex is present in one ubiquitously expressed form.9,10 The ␥-, ␣-, ␦- and ⑀-adaptins are the most individual subunits of the complexes and therefore they might be responsible for the compartment-specific binding of the adaptor complexes (see Membrane Binding). ␥-and␦- adaptin appear to mediate complex formation.14,17 Subunit interactions have been demonstrated between ␥- and ␴1- adaptins, ␥- and ␤1-adaptin and between ␤1- and both ␮- adaptins and ␮1-adaptin deficient cells do have reduced expression levels of ␴1-adaptin.13,23 This indicates that there are cooperative interactions between the subunits, which may be important for complex formation and function. ␤1- and ␤2-adaptins are essential for formation of the clathrin-basket surrounding the adaptor-coat. They bind clathrin at the flexible stalk and this binding is essential and sufficient for clathrin basket formation, whereas clathrin Figure 3 Assembly unit of clathrin baskets. Three heavy-chains binding to the N-terminal domains of ␥-and␣-adaptins is form a triskelion. Three light-chains (grey boxes) bind to the sec- only able to support polymerisation of the coat.11 ␤-adaptins tion of the heavy-chains proximal to the center. The N-terminus of are also able to bind the sorting signal sequences present in a heavy-chain forms a globular domain separated from the leg by the cytoplasmic tails of vesicle cargo proteins.24 ␤1-adaptin a flexible domain and projects inward. binds the microtubulie based plus-end directed KIF13A

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motor protein.25 The globular C-terminal ‘ear’ domains of Microscopic analysis of MPR/GGA colocalization and move- the ␥- and ␣-adaptins and of the ␤1- and ␤2-adaptins do ment of these vesicles to the cell periphery indicates that bind a number of proteins involved in vesicle formation and GGAs are involved in trans-Golgi network export of MPRs, fusion (see Relatives and Co-adaptor Proteins and Accessory a function thought to be fulfilled by AP-1.43 Interestingly in Non-coat Proteins). AP-1-deficient cells MPR46 accumulates in early-endosomes The predominant function of the ␮-adaptins appears to due to a block in endosome-to-trans-Golgi network trans- be the binding of sorting signal sequences in vesicle cargo port, indicating a function of AP-1 in retrograde transport proteins. Trimeric AP-1 complexes lacking the ␮1-adaptin of MPR46.13 GGAs and AP-1 might act in parallel export are not able to bind to membranes, indicating that ␮1-bind- pathways or between closely positioned exocytic compart- ing to membrane proteins, either cargo proteins or compartments. GGA2 might be involved in the Golgi to endosome ment-specific adaptor-complex receptors, contributes to the sorting of the sortilin receptors, homologues of the yeast membrane-binding affinity of AP-1. The crystal structure of Vps10p receptor which transports the vacuolar carboxypep- ␮2-adaptin complexed with tyrosine-based signal sequence tidase Y.45,46 peptides of the YxxQ motif has been resolved.26 The residues The AP-2 complex forms a mixed coat with ␤-arrestin, forming the YxxQ binding domain are conserved in all ␮- which binds to the cytoplasmic domain of activated ␤-adre- adaptins. Residues surrounding this motif also interact with nergic receptors and to clathrin.47 The monomeric AP-180 ␮2-adaptin which may contribute to binding affinities or (180 kDa) and its homologue CALM (clathrin assembly may even confer binding specificity.27 ␮-adaptins do also myeloid leukemia protein) form clathrin-coated vesicles in bind to leucine-based sorting signals.28 The polarized epi- vitro and display a higher affinity towards clathrin than AP- thelia specific ␮1B-adaptin mediates basolateral sorting of 1 or AP-2.48 It is expressed ubiquitously and exists in a neu- the low-density-lipoprotein receptor.18,29 Interestingly also ronal isoform. AP-180 also binds to AP-2 so they form the ␤-adaptins are able to bind to sorting signal sequences.24 mixed coats.49,50 This may increase the number of sorting-signal-sequences recognizable by the complex and thus increases the number MEMBRANE BINDING OF COAT PROTEINS of proteins sorted by the adaptor-complex. Binding of coat proteins to membranes is compartment- Sorting signal sequences in transport vesicle cargo pro- specific. The mechanisms mediating compartment speci- teins often contain a critical tyrosine and some are classified ficity are not known. Coat proteins are able to bind to lipo- as YxxQ or the NPXY motifs readily recognized by AP-2 somes devoid of integral membrane proteins, but protease endocytic vesicles. Interestingly the NPXY motif does not treatment of biological membranes reduces the binding bind to ␮2-adaptin, but is able to interact in vitro with the affinity of adaptors.51 terminal domain of the clathrin-heavy-chain.30,31 Others N-terminal sequences of ␥- and ␣-adaptins have been consist of a critical di-leucine motif or contain one leucine exchanged to study the influence of this domain for mem- residue and a second hydrophobic amino acid.32,33 There are brane targeting to the trans-Golgi network and the plasma also many sorting sequences which do not fall into these membrane respectively. Replacing 330 N-terminal amino classes and so far one can not predict from the cytoplasmic acids of ␣-adaptin by those of ␥-adaptin targeted the chim- tail sequences of a cargo protein to which of the adaptor- era to the trans-Golgi network. The adaptor complex con- complexes it will bind.33–37 sisted of the ␥/␣-chimera and ␤2, but contained ␮1 and ␴1.23 Nothing is known about the functions of the ␴-adaptins. A trimeric ␥-␤1-␴1 complex is formed in ‘knock-out’ cells of The ␴3A-adaptin does bind to the non-activated, non-phos- the ␮1A-adaptin, but this complex is not able to bind to the phorylated insulin-receptor-substrate 1 (IRS-1), however the trans-Golgi network.13 Therefore ␤-adaptins do not contrib- physiological significance of this interaction is not yet ute to the compartment-specific binding, but ␥ in concert known.38 with ␮1, and maybe also ␴1, are required. It is not known, whether the cargo proteins contribute to the membrane RELATIVES AND CO-ADAPTOR PROTEINS recruitment of AP-1, but ␮1-adaptin, which binds to sorting Recently three homologous proteins have been identified, signal motifs in the cargo proteins, is required for membrane in yeast and man, based on their sequence homology to the binding of the AP-1. The epithelia specific isoform ␮1B C-terminal ‘ear’ domain of ␥-adaptin.39–41 They are all local- forms the AP-1B complex, which mediates basolateral sort- ised to the late Golgi and are named GGA1 to 3 (Golgi-local- ing of the LDL-R in polarized epithelia.29 Interestingly AP- ised, ␥-adaptin homologue and ARF-binding proteins) 1, now also referred to as AP-1A, and the AP-1B complex do (Figures 1 and 4). They bind to the Golgi by directly inter- bind to different domains of the trans-Golgi network.52 AP-1 acting with ARF-1 and they bind the clathrin-heavy-chain.42 and clathrin have also been detected on immature secretory GGAs bind to the acidic-di-Leu based sorting signal granula. It is believed that they are involved in the removal sequences in the two mannose-6-phosphate receptors of membrane proteins.53,54 ␥-adaptin has also been detected MPR46 and MPR300, which mediate sorting of lysosomal on early endosomes in polarized cells, where it might be enzymes. Different duplets of acidic amino acids upstream involved in transport of proteins from apical endosomes to of the di-Leu sequence determine the specificity of the bind- basolateral endosomes.12 ␮1A-adaptin deficient and thus AP- ing to the GGA family members. All three bind to the 1-deficient cells have a block in endosome to trans-Golgi MPR300, but only GGA1 and GGA3 bind to the MPR46.43,44 network transport of the mannose-6-phosphate receptor

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indicating that the AP-1 might be directly involved in retro- chains bind to the heavy chains at their proximal segment. grade protein transport.13 These triskelions assemble forming hexagons and penta- Binding of the coat proteins AP-1, AP-3, AP-4, the three gons.8,11 Clathrin self-assembly is regulated by the light GGAs and the Golgi COP-I requires the small GTPase ARF- chains.63 1 (ADP-ribosylation factor 1; Figure 4). ARF-1 stimulates The clathrin heavy chain is ubiquitously expressed and a phospholipase D activity required for membrane binding of homologue is expressed mainly in skeletal muscle.64 There the COP-I, but AP-1 binding to the trans-Golgi network does exist two ubiquitously expressed clathrin light chains, LCa not require phospholipase D activity.8,39–41 The AP-1 com- and LCb, which are 60% identical. In the brain two splice plex has however been crosslinked in vivo to three different variants of LCa and one of LCb are formed.65 The expression membrane proteins, but they have not been characterized ratios of LCa and LCb vary, indicating functional differ- so far.55,56 ARF-1 appears to bind to ␥-adaptin.57 Formation ences.66 of phosphatidylinositol-3-phosphate at the trans-Golgi net- The N-terminus of the clathrin heavy chain has a propel- work appears to be required for AP-1 vesicle formation and ler configuration and binds to the consensus motif protein sorting.58 PI-3-P is able to enhance the binding of LLD/NLD in the C-terminal region of ␤-adaptins of AP-1 and sorting signals in the cytoplasmic domains of receptors with AP-2 and other coat-proteins.67 Although the ␤3-adaptin of the adaptor-complex.24 AP-3 binds clathrin in vitro, budding of AP-3 coated vesicles Binding of AP-2 to the plasma membrane does not require is clathrin-independent.68 Disassembly of the clathrin coat the presence of an ARF-family member, but does depend on involves the HSC70 (heat-shock-cognate 70) ATPase and the the presence of phosphatidylinositol-4,5-bisphosphate. Also co-chaperone auxilin, which in vitro bind to the clathrin ␤ 69–72 -arrestin and AP-180 do bind to PI-4,5-P2 via their ENTH light chains. domains and this interaction is essential for endocytosis.59,60 They also bind to 3-phosphorylated phosphatidylinositols.61 ACCESSORY NON-COAT PROTEINS Accessory proteins take part in the regulation of vesicle coat- CLATHRIN formation, vesicle budding and pinching-off of the vesicle as Clathrin assembles into a hexagonal lattice on membranes, well as uncoating. Vesicle budding and vesicle fusion require which during the process of vesicle formation, changes into interactions with and reorganization of the actin cytoskele- a basket made out of hexagons and pentagons on vesicles. ton.73 These proteins bind to the C-terminal globular The basic assembly unit is the clathrin triskelion, which is domains of ␥-or␣-adaptin and the ␤1 and ␤2-adaptins. composed by three clathrin heavy chains and three clathrin They are also able to bind clathrin and membrane lipids as light chains62 (Figure 3). The clathrin heavy chains trimerize the coat adaptins.74 with the C-termini in the center to form the triskelion. Each ␥-synergin binds to ␥-adaptin and is predicted to bind pro- ‘leg’ of the triskelion can be divided in a proximal segment, teins with an NPF motif.75 It is a homologue of eps15 which a distal segment and an N-terminal globular domain, which binds to ␣-adaptin.76 The binding site in ␣-adaptin is the is separated by a flexible domain. This linker allows the N- region aa 825–935 with W840 surrounded by charged amino terminal domain to project inwards. Three clathrin light acids. Also epsin, amphiphysin 1/2, AP180 and auxilin bind to the same region and all of these proteins are also able to bind to clathrin. All these contain the same DPF/W motif, which interacts with the W840 domain.77 Overexpression of the DPF/W protein is able to inhibit endocytosis.78 The fact that all these accessory proteins compete for the same binding site in ␣-adaptin indicates that they fullfill different functions at different steps in vesicle formation. Eps15 and its homologue Eps15R are the epidermal- growth-factor receptor kinase substrate 15. Eps15 contains two sequences called EH domain for eps15 homology domain. Each of them is formed by two helix-loop-helix EF- hand motifs. The EH domain binds to NPF motifs as in the adaptor protein AP180 and the phosphoinositide phosphatase synaptojanin.79 Eps15 phosphorylation by the EGF receptor is essential for EGF receptor endocytosis, but not for the constitutive endocytosis of the transferrin receptor.80 Proteins of the epsin-family contain three NPF motifs and bind to eps15.81 They contain a structural determinant

called ENTH domain for epsin-NH2-terminal homology domain. Eps15 and epsin are essential for endocytosis. Both Figure 4 Interaction of the GGA proteins with the acidic-di-leucine are phosphorylated during mitosis leading to a block in sorting motif of cargo-proteins, the small GTPase ARF-1, the endocytosis. Epsin and Eps15 cycle between the cytoplasm ␥ clathrin-heavy-chain and the -synergin. and the nucleoplasm. The ENTH domain is able to bind to

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the transcription factor promyelocyte leukemia zinc-finger remains bound to clathrin and may also assist in clathrin protein (PLZF). Eps15 and the homologue Eps15R are differ- re-assembly.69,70,72,97 ently expressed during organogenesis. Eps15 expression is high in proliferating neuroblasts, whereas Eps15R was found LIPIDS REGULATING VESICULAR PROTEIN in post mitotic cells. At later stages in development both TRANSPORT proteins were expressed in neuronal structures.82 Eps15R Membranes of a cell vary in their lipid composition and lip- was concentrated in the nucleus, whereas eps15 was local- ids are asymmetrically distributed between the two leaflets. ised to the cytoplasm and the plasma membrane.83 These Lipid metabolism is required for protein sorting and trans- data suggest that components of the endocytic machinery port. The inositol ring of phosphatidylinositol is mono- and are also involved in nuclear signalling. poly-phosphorylated at the 3-, 4- and 5-hydroxyl groups in Ese1 and Ese2 (EH domain and SH3 domain regulator of different combinations creating a number of phosphoinosit- endocytosis) proteins contain two EH domains, a central ides each fullfilling different functions in vesicle-mediated coiled-coil domain and five SH3 domains.84 Their splice vari- protein transport. Proteins bind to these lipids via different ants Ese1L and Ese2L contain in addition at their C-termini domains: PH, SH3, PTB, FYVE, C2 and ENTH. one DBL/PH domain and one C2 domain. DBL/PH domains PI-4,5-P2 at the plasma membrane is bound by the vesicle ␣ are found in exchange factors of small GTPases of the Rho coat proteins -adaptin of AP-2 via its N-terminal domain, ␤ family. Ese forms a complex with Eps15 and binds to dyna- AP180 via the ENTH-domain and -arrestin. Mutations in mins. Esel overexpression blocks endocytosis. Eps15 forms the PI-4,5-P2 binding site in AP-2 impairs membrane bind- oligomers through its central coiled-coil region suggesting ing of the heterotetrameric complex. Also accessory proteins that the Ese-Eps15 complex is multimeric. involved in the coordination of vesicle formation bind to Dynamin is a GTPase essential for endocytosis.85 It is PI-4,5-P2. These are amphiphysin and dynamin. Dynamin required for vesicle fission from the plasma membrane. It binds PI-4,5-P2 via its PH domain and binding stimulates oligomerizes into rings surrounding the neck of the budding GTPase activity together with SH3 domain containing proteins.98 Mutation of the PH domain inhibits endocytosis, vesicle. The mechanism by which this ring facilitates mem- which can be rescued by mutation of the PRD in dynamin brane fission is not known, however GTP hydrolysis is asso- which binds to SH3 domain containing proteins.99 Thus PI- ciated with a conformational change indicating a mechano- 4,5-P binding by dynamin appears to play a role in vesicle chemical process.86 It contains a PH domain (pleckstrin- 2 budding after dynamin has been recruited to the budding homology) which binds to phosphoinositides. It also has a vesicle. Synaptotagmin is a membrane protein of synaptic proline-arginine-rich domain, PRD, which binds to the SH3 vesicles, which binds to AP-2 and to PI-4,5-P via one of its domains of amphiphysin and endophilin. The isoform dyn- 2 two C2 domains. The second C2 domain binds Ca2ϩ and in amin 2 has been localised to the trans-Golgi network and is vitro modulates the affinity of the first C2 domain to PI-4,5- involved in trans-Golgi network to plasma membrane trans- P and PI-3,4,5-P .100 port.87–90 Interestingly dynamin 2 overexpression induces 2 3 The phosphoinositide phosphatase synaptojanin also apoptosis in a dynamin GTPase-dependent manner and binds to and dephosphorylates PI-4,5-P2. Thus it is expected because dynamin is able to interact with receptor-kinase sig- to be involved in vesicle uncoating and to be a negative nal transducers it is possible that dynamins are also involved regulator of endocytosis. This is supported by the obser- 91 in signalling pathways. vations that the uncoating ATPase HSC70 is not sufficient Amphiphysin isoforms 1 and 2 form heterodimers which to promote clathrin disassembly and that synaptojanin bind via their SH3 domains to the PSRPNR sequence in dyn- knock-out mice have an increased number of clathrin- amin. They also bind clathrin, synaptojanin and endophi- coated vesicles.71,101 lin.74 Isolated amphiphysin SH3 domains prevent dynein PI-4,5-P2 is formed by a PI-4-P 5-kinase associated with the oligomerisation. They recruit dynamin, but prevent its oligo- plasma membrane. Its substrate PI-4-P could be formed by 92,93 merisation until they dissociate. a plasma membrane associated PI 4-kinase, but it could also Endophilin is a lysophosphatidic acid acyltransferase be the PI-4-P present in dense-core secretory vesicles. PI-4- 94 required for efficient formation of synaptic vesicles. It adds P is generated by a PI 4-kinase associated with dense-core arachidonate to the sn-2 position in LPA leading to a mem- secretory vesicles and this enzyme is required for brane lipid with an larger axial diameter. This can impose a 102,103 exocytosis. Thus PI-4,5-P2 synthesis during exocytosis larger negative curvature at the neck of the clathrin-coated and its dephosphorylation during endocytosis could coordi- vesicle supporting the late stages in vesicle budding. nate these membrane transport steps. Phosphatidyinositol- Synaptojanin is a polyphosphoinositide phosphatase 3-phosphate is formed at the trans-Golgi network by the PI which acts on several compounds like phosphatidyl- 3-kinase Vps34p.104 Sorting of vacuolar/lysosomal proteins inositol-4,5-bisphosphate and phosphatidylinositol-3,4,5- requires PI 3-kinase activity.104–106 PI-3-P is present in early trisphosphate. It is enriched in nerve terminals and is endosomes and in the inner membranes of multivesicular expected to play a role in vesicle uncoating.95,96 endosomes.107 It is degraded in the vacuole/lysosome by a Auxilin is a co-chaperone of the clathrin uncoating HSC70 phosphatase.108 Proteins bind to PI-3-P via their FYVE- ATPase and stimulates its ATPase activity leading to the domain and FYVE-domains are sufficient to label PI-3-P con- release of the clathrin coat. While auxilin is released HSC70 taining membranes.107,109–111 The early endosome associated

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antigen EEAI binds PI-3-P via its FYVE-domain.112 EEA1 also factors enhancers and GDP/GTP exchangers. The Rab-GDP- binds to the GTPase Rab5 thus tethering the membranes of bound form is localised to the cytoplasm as a complex with two compartments for fusion. PI-3-P is converted into PI- GDI. At the membrane, interaction with GDF leads to dis-

3,5-P2 by the FYVE-domain PI-3-P 5-kinase Fab1p. Synthesis sociation of the GDI and GEF stimulates the GDP-GTP of Fab1p is induced by osmotic stress and Fab1p is involved exchange resulting in membrane bound Rab-GTP, where it in membrane homeostasis. PI-3,5-P2 is required for transport interacts with different effector proteins. Interaction of Rab- of multi-vesicular endosomes into the vacuolar lumen. GTP with a GAP leads to GTP hydrolysis and re-association Reduced levels of the lipid lead to fusion of these mem- of Rab-GDP with a GDI and relocalisation of the complex branes with the vacuolar membrane generating enlarged into the cytoplasm.119 Rab-GTP proteins are ubiquitously 113 vacuoles. PI-3,4,5-P3 is generated by PI-4,5-P2 3-kinases. expressed and tissue speciﬁc isoforms exist like Rab3A which

Coat proteins do bind to PI-4,5-P2 and to PI-3,4,5-P3, how- is only expressed in neurons or Rab17 which is only ever the precise role of the phosphoinositide-trisphosphate expressed in epithelial cells.120,121 Also the proteins reg- in endocytosis is not known.61 ulating the Rab-cycle come in isoforms. Rab proteins have many effectors like SNAREs, kinases and motor proteins. FUSION OF VESICLES WITH ORGANELLES Rabphilin binds to Rab3A and has structural homology to Binding of transport vesicles to their target organelle is synaptotagmin: two C2-like domains involved in Ca2ϩ and facilitated by the family of SNARE proteins (N-ethylmaleini- phospholipid binding. It inhibits GAP-induced GTP mide-sensitive factor attachment protein -SNAP-receptor: hydrolysis keeping Rab3A in the activated state. Rabphilin SNARE).114 The family has 21 members in yeast and over 35 stimulates ␣-actinin to cross-link actin filaments leading to in mammals.115 SNAREs of the vesicle termed v-SNARE and the formation of actin bundles. Reorganisation of the actin SNAREs of the target organelle termed t-SNARE associate cytoskeleton is associated with endo- and exocytosis. Rabki- with their membrane proximal helices to form a four-helix nesin-6 is a microtubuli-based motor protein localised to the bundle creating a coiled-coil domain (Figure 5). Formation Golgi, which binds to Rab6. Fusion of endosomes requires of these trans-SNARE complexes brings the two membranes Rab5, which is bound onto the membrane as a Rabaptin5- in close proximity. The coiled regions of the SNAREs contain Rabex5 complex (effector and GEF). Rab5 binds to the effec- either a critical arginine (R) or glutamine (Q) and tetrameric tor EEA1-early endosome associated antigen 1. EEA1 forms SNARE complexes always contain three Q-SNAREs and one a coiled-coil dimer. It contains two Rab5 binding sites at its R-SNARE. Therefore v- and t-SNAREs were reclassified in Q- N- and C-termini which are flanked by zinc-finger domains, SNAREs and R-SNAREs. All t-SNAREs are Q-SNAREs, whereas so called FYVE domains. One binds zinc and the C-terminal v-SNAREs are R-SNAREs or Q-SNAREs.116 binds PI-3-P.109,110,112,122–124 EEA1 is required for endosome SNAREs act at a late step in vesicular protein transport. fusion and Rab5 has to be present on both endosomes. Rab5 Proper and efficient targeting of vesicles to their destinations and EEA1 thus appear to tether the two membranes together requires proteins from the large Rab/Ypt family of small before SNARE proteins can form complexes. Interestingly GTPases. The first protein of this family was identified in EEA1 also binds to the SNARE syntaxin-6 via its C-terminus yeast (Ypt1) and the first mammalian homologue was involving a domain encompassing the Rab5 and the PI-3- named Rab for ras-like in rat brain.117,118 Rab proteins are P binding region. Thus the SNARE protein could cause the found on all membranes and cycle between the membranes, dissociation of the Rab5/EEA1 complex.125 Rabenosyn5 (Rab Rab-GTP state, and the cytoplasm, Rab-GDP state. The Rab- ϩ ‘enono’ Ϫ ‘to link’ in Greek – ϩ syntaxin) is a EEA1 homo- GTPase-cycle is influenced by several effector proteins: logue binding to Rab5, PI-3-P and the SNAREs syntaxin 4, GAPs: GTPase activating proteins, GDIs: GDP dissociation 6 and 13. Rabenosyn5 is required for lysosomal transport inhibitors, GDFs: GDI displacement factor, GEFs: exchange whereas EEA1 is not. Thus Rabenosyn5 and EEA1 may be involved in mediating the pairing of different SNAREs.126 The finding that Rab proteins have so many different Rab- effectors indicates that Rab-GTPases control many vesicular protein transport steps.

SUMMARY Vesicular protein transport involves the selective recruitment of cargo into the vesicles, controlled formation of the vesicle, partial uncoating and transport to the target membrane/organelle, binding to and fusion with the target membrane, followed by the exchange of the cargo molecules from the limited vesicular membrane domain to the mem- Figure 5 Formation of SNARE-complexes in trans during transport brane of the organelle. We are just beginning to understand vesicle docking and fusion with the membrane of the target how this complex pathway is regulated. Two important organelle. SNAREs are helical proteins, which bind to each with regulatory aspects we did not have space to cover are the their helices forming coiled-coil complexes. Each complex is formed role of protein phosphorylation as a regulatory switch and by four individual SNAREs. Here only two are drawn. the role of ubiquitinylation. The importance of ubiquitinyl-

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