Regulation

of intracellular

Jean Cruenberg European

Molecular

membrane

and Michael

Biology

Laboratory,

transport

J. Clague

Heidelberg,

Germany

A number of proteins that are necessary for membrane transport have been identified using cell-free assays and yeast genetics. Although our knowledge of transport mechanisms remains limited, common themes are clearly emerging. In particular, specific CTP-binding proteins appear to be involved, not only at all steps of membrane traffic but also at more than one check-point withln each step. The ordered sequence of events occurring during vesicle formation, targeting and fusion may be regulated in a stepwise manner by specific CTP-dependent switches, which act as modular elements of the transport mechanism.

Current

Opinion

in Cell

Biology

Introduction

Several proteins that are required for vesicular transport or homotypic fusion ha\v now hcen identified. These include cxmponcnts of the proteinacews cwt on so1lle transport \wicles. regulator elements hekqing to GTP-binding protein Eunilies Lund the N-~th)~lmaleimicle~ sensitive fusion protein ( NSF). NSF. mhich W;IS isolntccl using an itt rdttm xsay that me;Lsures intrn-Golgi ttxnsport, appexs to he LIS~CI during se\vral tmisport steps. Together with the soluble NSF-nssoci:tted proteins ( s(SNAP is Sccl7 in !‘east ). NSF fomis ;I protein complex that ~KI!~ regulate all intracellular fusion events [ 5.1, Accordingly, SeclX. the yeast equivnlcnt of NSF, is required for multiple tr:lnsport steps in !.east [ 61, Other proteins that appear to be involved in specitic tl’;insport steps, e.g. co;lt components :mcl GTP-binding proteins, can he grouped hy analog), of sec~wnce :tnd function into larger t:,lmilies. A further hmily ws recentI!. revealed by sequence homology hetwecn yeast genes encoding proteins required for spccitic steps in clitierent p:ithways [ 7.1. Although rel:ltivel\~ few components have been identified, the existing &it:1 argue that conserved designs are being used in the mechanisms of different steps of membrane transport. focuses proteins

on

the in

regulator membrane

role of transport,

4:593-599

artzi of research is developing rapidly. Membmne transport is ;L relativel~~ recent addition to the long list of cellular processes kno\T~i to be dependent on members of the GTP-hinding protein super-fximily [8]. Originally, two yeast genes, 1’PTI and SW~. whose products xe required ;It specilic steps of the hiosynthctic p&way, were shonn to encode small GTPhinding proteins homologous to the t-us proto-oncogene product [9]. This famiI!. of small (2ct30 kD ) monomeric GTP-binding proteins has since been csp;mded and cli\~ersifetl, such that it now includes the Rx, YPTl ‘Sec-L’Rah, Rho and ARF/‘Sar subf:umilies [ lO,ll]. Of theseit appears that members of the \‘PTl 5ectW-1 and the ARF Sar suhfrunilics are imVolved in nienihrme transport, and ma!’ be required for different processes during vesicle formation. co;tt :membly, targeting :tnd. possibly, fusitjn. Finall!,. ;I major development has been the linding that trimcric G-proteins ( GaPy 1, which mediate tr;insmembrane signalling by homiones xncl light at the c;Toplasmic face of the plasma membrane [8], also h:i\,e :t role in membrane tr:tic.

Since the 0rigin:il proposd by IUacle ( re\ie\vccl in [ I ] ), it has gencrxll!~ hccn accepted that memhranc transport in the secretory pxh\\x); is mediated by small. short-lived vesicles th:lt bud from one compartnwnt and are then txgeted to the nest comp:trtment in the pathway. with which the!. r:ipiclly fi~sc. In nclciition to hcing connected hy this \vctori:d membrane tlow. se\~eral lines of evidence indicutc that the conymtnwnts involved in membrane trmsport ;ire highI)* plastic and cm form dyn:imic net. works, in ~1 process that presum:lhly involutes homot\l%c fusion and fission c\ents [7-i ]

This review GTPhincling

1992,

Dissection

of vesicular

transport

A major hottlencck in stucl!.ing the hiochemistn of vesicul:tr transport has been the cliticul~ encountered in the isolation of vesicular intermediates. The best charxxericed txunple of constituti\~e transport vesicles 3re the clathrin-coated vesicles, which mecli:ite endoc?Wsis from the plasma membKune toR’3rds endosomcs and tmnsport from the butts-Golgi network (TGN) to endosomes [ 121. Other transport vesicles have also been purilied and char:icterized [ 13-171. Of fiinclnmental imporUnce to our understanding of \resicular transport is the ohsenxtion that, during transfer from the endopklsmic reticulum (ER) to the Golgi in yeast. cliff+rent gene products go\‘ern the formation and consumption of transport vesicles (see Schekman, this issue, pp 587-592 ). More recentI),, fractions containing

different ~1s this Abbreviations

CRF-guanine

ARF-ATP-ribosylation nucleotide-releasing

factor; factor;

@

ER-endoplasmic reticulum; NSF N-ethylmaleimide-sensitive

Current

Biology

Ltd

ISSN

GAP-CTPase-activating fusion protein;

0955-0674

protein; TCN--frans-Golgi

network

593

594

Membranes

these presumptive transport vesicles have been prepared and used in cell-free transport assays [ 18,19:,20]. In l&-o studies have shown that the formation of transport vesicles during transfer from the ER to Golgi in yeast depends on Sarlp, a small GTP-binding protein 1211 and SeclZp, a membrane protein [ 19*]. The formation of these vesicles apparently requires direct interactions between both proteins [ 22*,23*]. GTPyS, a non-hydrolyzable analogue of GTP that inhibits essentially ail membrane transport steps, also inhibits transfer from the ER to Golgi in yeast. Sarlp is likely to be a target of GTIyS, as permeabilized Secl2B cells were rescued by bacterially expressed Sarlp, but not by Sarlp pretreated with GTPyS [23*]. Attachment of the transport vesicles to their acceptor membranes required both Yptlp [ 19*,20], a prototype member of the Sec4/YPTl/Rab family, and Secl8p [ 19*], the yeast homologue of NSF. These experiments suggest that Sarlp and Yptlp, which belong to different subfamilies of small GTP-binding proteins, are required for vesicle formation and targeting/fusion, respectively, during the same step of vesicular transport. In eukaryotes, a similar genetic and/or biochemical dissection of the formation and consumption of transport vesicles has not been possible. One mutation (shibire) has been discovered in Drosophila that blocks coated vesicle formation at the plasma membrane [24]. The gene encodes a protein homologous to dynamin 125,261, a microtubule-based motor [27]. The functional significance of this observation remains to be elucidated, as many lines of evidence argue against the involvement of microtubules during internalization. Using different biochemical assays in mammalian cells, two groups have studied the formation of clathrin-coated vesicles at the plasma membrane. Both imagination and budding required ATP and cytosol in semi-intact cells [ 281, whereas only the budding step was ATP- and cytosol-dependent when plasma membranes were immobilized on a substratum [29]. This discrepancy presumably reflects differences in the protocols or in the stages of pit invagination measured in the assays. An in rWo assay has recently been established that measures the fusion of purified plasma membrane-derived coated vesicles with endosomes [ 301. Rab proteins In mammalian cells, each Rab protein that has been iocakzed by immunocytochemistry is distributed to the cy toplasmic face of specific subceiiular compartments [9] (Fig. 1). This has strongly reinforced the view that Rabs are involved in specific steps of membrane transport. A recent study demonstrates that the specific association of Rab proteins with their correct intracellular membranes is mediated by a signal present within the carboxy-terminal hypervariable domain [31-l. No membrane receptor for this signal has yet been identified. Membrane attachment itself requires isoprenylation of a carboxy-terminal cysteine. For both Yptlp and Sec4p in yeast, this step depends on Bet2p, a putative component of a protein prenyl transferase [32]. In mammalian cells, different protein prenyl transferases acting on membranes of the Rab subfamily have been identified or purified [ 33-361. It

is important to establish whether transferases, like their substrates, are part of a larger family. In a mammalian cell line, the Rab5 protein, which is found in early endosomes and the plasma membrane, is required for the homotypic fusion of early endosomes in rWo [37*]. Neither mutation of Rab5 in the GTPbinding domain (Rab5Ile133) nor deletion from Rab5 of the cysteine motif required for membrane association supported fusion in this assay. Overexpression of wildtype Rab5 irz lkjo stimulated endocytosis and increased the size of early endosomes, whereas overexpression of Rab5Ile133 inhibited endocytosis and caused early endosomes to fragment [38]. In addition to Rab5, Rab4 is also found in early endosomes [39], and experiments in Lu’zrosuggest that it is involved in transport between the plasma membrane and early endosomes, possibly at the recycling step (P van der Sluijs, M Hull, P Webster, M Goud, I Meliman, unpublished data). Rab3a is implicated in synaptic vesicle exocytosis [40], as well as amylase release from pancreatic acini [41]. Finally, inhibition with specific antibodies has indicated that the Rablb protein, which is the mammalian homologue of yeast Yptlp, is required both during transfer from the ER to the Golgi and within the Golgi [42-l. Taken together, these studies indicate that proteins of the Secii/YPTljRab family are necessary both for vesicular transport and for homotypic fusion events, consistent with their proposed involvement in targeting [43]. It remains to be seen whether ali Rab proteins are functionaiiy equivalent at different steps, and whether isoforms of the same Rab [lo] reflect redundancies or a high degree of specialization. Regulation hydrolysis

of nucleotide

exchange

and

All small GTP-binding proteins are believed to undergo a conformational switch upon exchange of GDP for GTP, which is then reversed upon GTP hydrolysis. This switch can then be used to introduce vectoriakty into a celiular process via the regulation of GDPGTP exchange and GTP hydrolysis [431. Under physiological conditions, nucleotide exchange and hydrolysis may be contingent on interactions with both a guanine nucieotide-releas ing factor (GRF) and a GTPase-activating protein (GAP) [9,331. A cytosoiic 275 kD GAP active on Rab3a, but not ,RabZ, has recently been described [ 441, and two cytosolit Yptlp/Rabl GAPS have been partially purified [45]. In addition, two GRFs have been identified, one acting on Rab3a and the other on more than one small GTPbinding protein, including Ras [46,47]. It is essential to establish to what extent GRFs and GAPS are shared by different small GTP-binding proteins, and whether they are associated with specific subcellular compartments. Further complexity is added by two other possible means of regulation. First, proteins that inhibit the dissociation of GDP have been identified, in particular, a cytosolic inhibitory protein, which specifically recognizes the GDP form of Rab3a and promotes its dissociation from synaptic vesicles (see [33] for review). Rab3a dissociates from membranes after stimulation of synaptic vesicle exocy-

Regulation

of intracellular

membrane

transport

Cruenberg

and Clague

Extracellular

Sec4p

\

Constitutive \

\ 1

(yeast) ECV

\

TCN C

Golgi complex

Ly5osome

Rough ER

Fig. 1. Newly

synthesized proteins leaving the endoplasmic reticulum (ER) are transported sequentially through a pre-Colgi compartment, the Colgi complex and the trans-Golgi network (TGN) [4]. After sorting in the TCN, they are routed to the plasma membrane (via the constitutive pathway or the regulated pathway in some cells) or to endosomes 1811. Endocytic stations comprise peripheral early endosomes (EE), which may be organized in a dynamic reticulum via fusion/fission events I21, perinuclear late endosomes (LE) and lysosomes. Endosomal carrier vesicles (ECV), moving on microtubules, may mediate transport between EE and LE. Whether these vesicles are specialized transport vesicles 1821 or intermediates in a maturation process [831 is not clear. Small CTP-binding proteins that have been localized to specific compartments are indicated, Steps that are sensitive to [AIF41and the py-subunits of heteromeric C proteins are shown. Inhibition by GTPyS is not illustrated, as all steps are essentially CTPyS-sensitive 191 and GTPyS does not discriminate between GTP-binding proteins.

tosis [40]. Whether dissociation occurs in response to GTP hydrolysis is not known. Another fundamental level of control is suggested by recent genetic studies in Drosophila. These data imply that both a protein homologous to Ras GAP and a nucleotide diphosphate kinase regulate the same GTP-controlled developmental process [@I. 1~ zlifro, M-GDP can serve as a substrate for nucleotide diphosphate kinase [@I. H

Heterotrimeric

G proteins

AIMindication that heterotrimeric G proteins (GaPy) are involved in membrane transport came from the observation that several steps of membrane transport are sensitive to [A&~] - in mammalian cells (Fig. 1). This reagent does not affect any small GTP-binding protein

595

5%

Membranes

that has been tested, but it activates heterotrimeric G JXOteins [50]. Evidence was also obtained by&showing that over-expression of Garij retards the secretion of a constitutively secreted proteoglyran [ 511. This effect can be ahrogated by treatment with pertussis toxin. which specifically ribosylates Gai subunits. The formation of J>ost-TGN secretory vesicles in l+tt-o can be inhibited by [AIF,] -, and stimulated by purified G& subunits or by pertussis toxin-mediated ribo.sylation of Gai3 ( [ 571; F Barr. A Jqte, W Huttner, unpublished data). The sensiti\it\. to pertussis toxin implies that the J3rocess invol\.es interaction between the heterotrimer and a nucleoticlc exchange factor. These exchange factors ha1.e been well described for signal transduction at the cell surface: the best characterized examples correspond to receptors that t\picnll!. span the plasma membrane se\~n times. Vesicle

coat assembly/disassembly

The coat composition of two ty~s of transJ,ort \.esicles has now been well characterized ( [ 121; see Kreis. this issue, pp 60!+615); these are the clathrin-coated \.esicles (involved in transport from the plasma membrane to cndosomes and TGN to endosomes), and the non-clathrincoated vesicles (involved in intra-Go@ transport). Of fundamental interest is the obsenation that a similar grouJ> of proteins may form both npes of coats. In particular pCOP, a component of the non-clathrin coat [ 53). shares homology with fi-adaptin [5-4*], a comJ>onrnt of the clathrin coat. It is generally accepted that at each round of membrane transport, the components of the clathrin coat are recruited from the cytosol onto the membrane during vesicle formation and then released into the c\~)sol after budding [ 121. Studies using GTPyS and br~feklin A. a drug that inhibits transport in the biosynthetic pathn-a). [3], suggest that the coat of non-cluthrin-coats vesicles can also cycle between qtosol and membranes [ 55-5’1.

ing. tithough it is not clear at JTresent whether trimeric G J3roteins are directI!, involved in the control of co;lt assembly, or whether they ha\~ other functions in menlbrane transport. these studies su,ggest that monomeric and trimeric G-proteins interact in some ~1)~. This has alread!. been dem~~nstrated in transmemlX1ne signalling IX. unccqlinq the opening of the K + ch:innel from the sdmulation oi- muscarinic (hl.! ) cholinqgic receptors. via an interaction in\.ol\ing Ras and Kas GAP 1651. Cell cycle

regulation

of membrane

transport

During mitosis, bic~s~~ntheticand cncloc!?-ric m~mhran~ transJ3ort is arrested in mammalian cells [ 66 1. Irt c?ttn studies ha\*e suggestccl that this inhibition ma!’ he mecliated 1~7the cell-c?.cle control protein kinase cdc2 [6’1. which ‘is rissociatecl \lith c?~c%nl3[ 681. :I c~omJ4cs th:lt regulates the G) XI transition. Mitotic c?tosc)l can also inhibit the in\q$i:itic~n of co:itccl pits in hrokcn cells (671. Two Rab J>rotc’ins. KabI:i :incl Kah-ia. i\‘erc rc’centlv reJ3orteJ to be J~hosJ~lio~~l;itcd itr I,ir~) during mitosis and irr r*iftu I,!, the cdc2 kinasc. \\hcrcas c)thcI Rab proteins \vere not [TO], This suggests th:it R;II-JI:L and Rab-ta may he irn~oh4 in mitotic mcmI~T;Inc’ tr:lRic arrest. An interesting feature is that both f J>rotein of unknvnn function. nhich co-fractionates \\ith the Golgi. n3s recentI!. reported to IX J~;~lmito\~latecl during mitosis and during brefekiin A-mediated inhibition of hccrction 173-l. How does membrane

fusion

proceed?

Recent studies have re\.ealed that :I J3rotein of the ADPribosylation factor (ARF) subfami$. ma!. itself be a com ponent of the coat of non-c1athriil-coated \.esicles [ 5H*] and may be implicated in both transport from the ER to the Golgi [ 571 and in homot\pic endosome fllsion [ 601. ARF proteins are small GTP-binding proteins \vith sonic’ homology to SarlJl, originall). identitied as a co-factor in the cholera toxin-mediated riboh>Mon of heterotrimeric G, proteins [61-63]. ARF purified from qtosol is GDPbound [62] and only attaches to membranes when it is in the GTP state. Thus, Serafini et rrl. [5X*] have J>ostulated that, during intra-Golgi transport, a GDP4TP exchange protein regulates ARF association with Golgi membranes, and that coat disassembly after vesicle targeting is controlled by GTP hydrolysis via a GAP protein localized to the acceptor membrane.

Although man!’ ccll~frce :IXI!Y mc:Isure the c)c‘c.urrcnce of fusion. little is kno\vn about the mechanisms imy )I\.ccl. Studies \\itli :irtiticial bila).ers h3.e cstablishccl th:it close contact beM.ccn bila!~ers ( < 20 ,A) is cnergetic:lll)~ \‘cr?’ uiifa\~c~urable. due tcj 3 strong rrpulsi\~e ft Kc ( h~drXion reJ3ulsion ). 1vhic.h is J3rc)habl>~ cleril.4 from the ordering of \\.;itcf molecules at the bila!.er surface [ ‘-41. For fusion to occur. this hydration repulsion must be o\.ercomc or circumvented. Recent studies using bila!,ers suJ3~3orted()n mica plates suggest that one means b!. n.hich the rt’J7ul sion can be o\crcome is the generation of 3 long~rang:e :ittracti\re force. \\hen the bila!,cr surface is made more h!~clroJ3hc )bic, for esam~3le, 1~~.the insertion of an aimphiJ3athic molecule [ ‘51. Conlirmation of this obsen;ltic)n hl. other techniques is now of JXimq~ imJ3ort;mce.

Heterotrimeric G proteins may also be involved in controlling coat assembly/disassembly. Thus, [AIF,] -, which may act only on trimeric G J3roteins [50], inhibits the dissociation of both P-COP [ 56,641 and y-adaptin [57]. The addition of purified by-subunits of heterotrimeric G protein inhibits the GTPyS-stimulated association of ARF and P-COP with Golgi membranes [64]. These ohsemtions may provide a functional link for the relationshil-, between ARF and Gapy, which has always been intrigu-

For the fusion of biological membranes, it has been SKI+’ gested that two meml~mes can be bridged 1~).speclhc oligomerization of membrane JXoteins. thereby form ing a collar or J3ore structure [76,77], These models then J>redict that lipid flow is initiated along the extended hydrophobic surfaces of the proteins. The best characterized exanmmplesof proteins that induce membrane-mcmhmne fusion arc \.irnl sJ3ike tr3~smemhmne gl)coproteins, in particular the intluenzn hemag-

Regulation

glutinin. During influenza infection, the virus is endocytosed, the low endosomal pH causing the viral envelope to fuse with the endosomal membrane. In this process, the hemagglutinin undergoes a low-pH triggered confonnational change, thereby exposing a previousl) buried amphipathic a-helix of approximately 20 amino acids. A similar amphipathic helix has been found in many other viraI spike protein sequences. Interestingly, the same motif has recently been identified in the sequence of PH-30, a sperm surface protein implicated in spermqg fusion [ 781. Intracellular fusion events, however, are under the control of different mechanisms and are expected to reuse components. If, by analogy with viral proteins, components unfold in response to a fusion trigger, they should then refold, perhaps via an energy dependent mechanism. Conclusions Several components of membrane transport mechanisms have been identified, for example, NSF and coat proteins. In this review, we have emphasized the regulatoq role of GTP-binding proteins. Recent data suggest that monomeric GTP-binding proteins of the Sec-r/YPTl ‘Rab and ARF/Sar subfamilies may act as modular elements of the basic mechanism within each transport step. Heten)trimeric G proteins may IX expected to prtn!ide a mc%msof coupling intracellular signallin~‘regulntion with this basic transport mechanism. However, the precise functions of GTP-binding proteins in membrane transport are far from clear. Currently, we can assume no paradigm for their mechanism of action beyond the notion of a conform:itional switch dependent on the nucleotide state. ClearI). then, the nucleotide state of these pn>teins should be determined at each stage of the process that they control, 3s well as the requirement for factors that promote exchange or hydrolysis.

of intracellular

PELII,L\I HRB: 67:449-t51.

4.

~MiwnimI, Slhto~sK: Cdl

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Cruenberg and Clague

transport

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Ii

Although some aspects of membrane transport mechanisms are now hcing studied in detail at the molecular level, essential components of these processes still remain to he identilied an&or characterized. Suiprisingly little is known ahou~ the function of lipids, although a clear relationship has heen obsen~l in yeast hemeen secretoq’ mcmhranc transport and lipid biosynthesis (?9,80*]. Moreover, sonic’ activities can he predicted for which proteins ha\Pe not yet been clearI!, assigned. These incluck two categories of specifc membrane receptors that are required for the docking of cytosolic factors, and for memhrruic-membrane recognition. Finall!., the catalysts of the fusion process itself remain to be identitied.

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Pathway

1)s I’irw.

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ated with 8B:63 1363 in

Early I-.

Endosomcs.

f’rix

1992.

in prc’w

A. GOI’I) 1%. Protein Rab-1 is Associ.Urll .-litrll .Scr I ‘.?A 1991, h.

7‘;\\‘l~rlAS

Fiwiw \‘os .\loij,wr~ G. Si’i~ iov TC. J;vis binding Protcin Dissociates from Synaptic Exoq-tosis. ,Vrrlrrrv 1991~ 3-19:‘9-XI.

R. A Small

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Golgi

In semi

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Pcptide R&&se :lctrcl ,%I

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