receptor-mediated endocytosis. The development of cellfree assays which directly measure coated pit assembly, invagination, receptor-sorting and coatcd vcsicle budding has recently provided an important first step towards addressing these issues and elucidating the mechanisms involved in the complex overall process of reccptor-mediated endocytosis.

Summary Keceptor-mediated endocytosis occurs via clathrincoated pits and is therefore coupled to the dynamic cycle of assembly and disassembly of the coat constituents. These coat proteins comprise part, but certainly not all, of the machinery involved in the recognition of membrane receptors and their selective packaging into transport vesicles for internalization. Despite considerable knowledge about the biochemistry of coated vesicles and purified coat proteins, little is known about the mechanisms of coated pit assembly, receptor-sorting and coated vesicle formation. Cell-free assays which faithfully reconstitute these events provide powerful new tools with which to elucidate the overall mechanism of receptormediated endocytosis. Introduction The efficient internalization of cell surface receptor-ligand complexcs occurs via a complex, multi-step process termed receptor-mediated endocytosis. Cell surface receptors carrying ‘endocytosis’ signals on lheir cytoplasmic tails are conccntrated in specialized coated pit regions of the cell surface(’).Coated pits invaginate and pinch off forming coated vesicles which carry receptor-ligand coniplexes into the cell. Thc coat is rapidly shed from the transport vesicle releasing its constituents for repeated rounds of coated pit assembly, invagination and budding. Coated vesicles, critical intermediates in this overall process. can be isolated in large yield from a variety of sources. Coat constituents, which arc likely to play a role in the selective packaging of membrane proteins into coated vesicles, arc rcadily purified. Extensive research efforts over the past fifteen years have providcd considerable insight into the structure of the coat and the biochemistry of coat constituents(2-6),.These results, derived from studies on the interactions of purified coat constituents with theinselves and with stripped vesiclc membranes, will be briefly reviewed hcrc. In the absence of direct assays for endocytosis, the role of these coat constilucnts in coated pit assembly, receptorsorting and coated vesicle formation could only be inferred from the nature of their interactions in in I’itro coat assembly assays. As a result, many mcchanistic questions remain to be answered regarding the dynamic cycle of coat disassembly which is intimately coupled to the process of

Clathrin, the Assembly Unit of the Coat Clathrin triskelions are three-legged structures consisting of three heavy chains (HC, -190 kD) and three tightly associated light chains of two classes, LCa (-23 kD) and LCb (-27 kD). The triskelion can be structurally and functionally divided into proximal and distal leg sections separated by a flexible hinge region and a globular ‘terminal domain‘ (Fig. I). Light chain-heavy chain interactions occur over an extended region of the molecule involving the entire proximal section of the heavy chain leg and the middle 1 /3 o f the light hai in'^.^'. Clathrin triskelions are the assembly units of the polygonal lattice on the surface of coated pits and coated vesicles. Proteolytic digestion cleaves the clathrin HC along the distal section of the leg into ‘truncated’ triskelions and terminal domains. The light chains are digested into small peptides which are released. Intact, truncated and light chain-depleted triskelions can all spontaneously self-assemble to form ‘cages’ in Ca”+-coiitaining low pH and low ionic strength buffers. indicating that under these conditions neither the light chains nor the terminal domains are required for cage a s ~ e i n b l y (The ~ ~ ~stnicture ~. of the cage in vitreous ice has been examined by eleclron microscopy (scc ref. 2 and refercnccs therein) . A triskelion lies at each vertex of the asseinbled cage. its leg spanning two edges of the polygon. Each edge of the polygonal lattice is therefore comprised of two proximal and two distal leg segments. The terminal domains are oriented toward the intcrior o f the cage. The light chains are bound to clathrin along the edges of the lattice and are presumably accessible to interaction with cytosolic factors. Based o n their position in the coat, their possession of Ca2+binding sites and phosphorylation sites, their ability to inhibit clathrin assembly and their interaction with uncoating ATPase (scc below), it has been suggested that light chains participate in regulating clathrin assembly and disassembly in vivo(7).

-

AP’s: Their Potential Roles in Coat Assembly, Membrane Attachment and Receptor Sorting The second major class of coat proteins have been variously lcrmed assembly proteins, clathrin associated proteins and more recently adaptor protein^(^,^)). As their exact function in viva has yct to be determined. I will conveniently refer to these proteins as ‘APs’. Two structurally related classes of APs are present in most cell types (Fig. 1). Both are heterotetrameric molecules. AP1 consists of two distinct -100-110 kD subunits, termed y and p’ adaptins, and two smaller subunits of -47 kD and -19 kD. Similarly, AP2 consists of -100-1 10 kD a and j3 adaptins, and two smaller subunits of -50 kD and -17 kD. Sequence data has shown that p and p’

Clathrin triskelion

AP complex

flexible

clathrin HC 190 kD

,

‘adaptins’ 115-100 kD

-. 5 .

50 or 47 kD subunit *-

trypsin

1 +

truncated triskelion

clathin LCs -25 kD

19 or 17 kD subunit

I

elastase

e e c terminal domains

trunk

heads

Fig. 1. Summary of the structure of clathrin and AP complexes, the major coat constituents of clathrin-coated vesicles. For exccllent. more coniprehensivc reviews on clathrin and AP structure see re[. 2,5-7.

adaptins are closely related to each other(”), as arc a and y adaptins“2J,p and p’ adaptins are quite divergent from a and y adaptins. ’There is also considerable sequence identity between the SO and 47 kD subunits and between the 17 and 19 k D subunits(I3). By rotary shadowing, AP2 appears as barrel-shaped molecules (termed ‘trunks’) with two appendages (termed ‘heads’) attached via a protease-sensitive flexible stalk[l3).The ‘trunk’ is comprised of the N-terminal -two thirds of the two adaptin subunits in tight association with the two smaller subunits. The two ‘heads’ correspond to the C-terminal third of either the a and p or y and p’ adaptins (see refs 5, 6 and references therein). Although A H molecules have not been examined, their sequence similarities with AP2 suggest a similar structure. By immunofluorescence. AP1 colocalizcs with Gol,‘wassociated coated pits, while AP2 colocalizes with plasma membrane-associated coated pits (see refs. 10: 5 and references therein). In addition to API and AP2, neurons also exprcss two additional clathrin associated proteins, AP3 (also termed AP180 and NP185)(lS)and ‘auxilin’(16).Both are monomeric proteins which bind and coasscmblc with clathrin triske6, in vitm. Their neuron-specific functions are unknown.

APs mediate clathrin assembly in the absence of membranes in vitro APs were originally named ‘assembly’ based on their ability to stimulate clathrin assembly under physiological conditions of pH, ionic strength and in the absence of divalent cations(17). APs co-assemble with clathrin into structures termed ‘coats’ which are smaller and more uniform in size than ’cages’(17).APs are found in the intcrior of the clathrin latticc, interacting with the terminal domains and pmjectiiig towards the vesicle rncmbrane (see ref. 2 and references therein). APs also bind to preassembled clathrin cages(18.1‘~~. Both the trunk and head domains of .4Ps are required for assembly activily and for high affinity ( h - 1 0 0 nM) interactions with triskelions and assembled cages(20). Clathrin binding appears to occur through the !3 or p’ subunit of AP’s“*l, although both a and p subunits are required for assembly activity(*l’. APs bind tightly to cages consisting of intact triskelions and less tightly to cages assembled from truncated triskelions(19J, suggesting that they interact with both domains of the clathrin HC.

APs may mediate clathrin binding to membranes in vitro Structural analysis of coaled vesicles has shown that APs are

positioned bctwecn thc clathrin lattice and the vesiclc mcmbrane(2).In addition, biochemical evidence suggests that APs may mediate clathrin binding to membranes. Clathrin can be selectively stripped from vesicle membranes by low ionic strength washes at high pH (pH 9.0). Under these conditions, 70-80% of the clathrin is released while most of the APs remain associated with the membrane. Tsolated clathrin binds to pH-stripped membranes with high affinity (3- 10 nM)(”). In contrast, clathrin binding is losl when the -100 kD AP subunits are removed either by elastase digestion or by more stringent wash conditions (pH 11 or 0.5 M Tris,pH 7). Purified APs also bind with high affiriiiy (30-100 nM) to rnernbranes which have been stripped of coat proteins by sodium carbonate extraction(23). The ability of APs to bind both membranes and clathrin is consistent with their proposed role in mediating clathrin-membrane interactions. In support of this, Virshup and Bennett(’3) showed that APs restored clathrin binding to carbonate-extracted crude bovine brain membranes. However, neither the stoichiometry of association, the nature of the structures formed, nor the identity of the membranes involved were examined. Although these data suggest that APs mediate clalhrin binding to membranes. there are several inconsistencies which remain unresolved. For example. intact and truncated triskelions bind to pH-stripped membranes with the same affinity‘?’) suggesting that terminal domains are not required. This conclusion appears inconsistent with the above-nientioned studies on coat structurei2! and on AP-cage interactionsi19)which indicated direct AP-terminal domain interactions. In addition, the affinity of clathrin-membrane binding (10 nM) is approximately 10-fold higher than that of clathrin-AP interactions. These two results suggest either that self-assembly with the residual clathrin present on pHstripped membranes might contribute, at leas1 in pari, to the observed binding or that other factors might be involved in clathrin binding to membranes.

APs interact directly with cytoplasmic tails of receptors in vitro Kcccnt evidence suggests that APs may interact directly with ’endocytosis’ signals on the cytoplasmic domains of rcccptors(24,25).Affinity matrices were constructed using fusion proteins containing the cytoplasmic tail of either the LDL (low density lipoprotein) receptor, found in plasma membrane-associated coated pits. or the CI-MW (cation-independent mannose-6-phosphate) receptor, found in both Golgi and plasma membrane-associated coated pits. Purified, [‘2sTllabelled AP2 complexes were retained on the LDI, receptor-tail column and eluted with 1.O M Tris, while purified 1”51]labelled API complexes failed to bind. In contrasi, both A P l and AP2 complexes were retained on the CI-M6Preceptor tail column. Competition studies suggested that the t.wo APs bind to distinct sites on the CI-M6P-receptor tail(2s),. Although this suggests that APs can interact directly with receptor lails in vitm, it remains to be seen whether other factors are involved in receptor-coat interactions in viw. The domain or subunits of APs involved in membrane and/or receptor binding have not been identified. However, it has been suggested that AP-receptor interactions occur through

the stalk and head regions of thc adaptin polypeptides. Thls hypothesis is based on primary sequence data for several adaptiiis which demonstrates that the greatest sequence diversity occurs in this region“’,”).

Models for AP Function in vivo Receptor-mediated endocytosis of transferrin is inhibited following inicroinjection of monoclonal antibodies specific for a-adaptins(”), demonstating the functional importance of APs in vivo. Although the specific functiods) of APs in L%IO has yet to be established. a working model for their role in coated vesicle-mediated transport has emerged rroin Lhe large amount of information on the biochemical and functional properties of APs in vitro . APs are proposed to initiatc coated pit assembly on the membrane, to regulate coated pit growth and to mediate receptor-sorting into coated pits by binding dircctly or indirectly to the cytoplasmic tails of receptor molecules. This proposal is consistent with the following properties of APs, which have been discussed above. I ) In the absence of membranes, APs mediate clathrin assembly and regulate the size of coats. 2) APs bind both clathrin and membranes and may mediate clathrin binding to membranes. 3) AP1 and AP2 have distinct intracellular localizations and exhibit sequence diversity which is consistent with a role in specific sorting events. 4) APs can interact directly with cytoplasmic receptor tails. There are other properties of APs, however. whose functional significance remains to be determined. For example, APs bind to preassembled clathrin cages with high affinity. AP2 specifically self-aggregates with high affinity (Kd-lO-* M) in the absence of clalhrin under coat assembly cond i t i o n ~ ( ~ ~ In , * ~addition, ). there are AP-associated kinase activities (see below)which have no known or ascribed function. Therefore, other models for the role of APs in t!ivo consistent with their known properties cannot be ruled out. For example, APs may enter planar clathrin lattices only after assembly is initiated and receptors are recruited. In this model, by regulating coat curvature and inkractirig with both clathrin and receptors, they may operate (via their kinase activities?) as switches which trigger coated pit invagination only after confirming that the clathrin lattices are fully loaded with receptors. Altcmatively or additionally, APs may serve as targeting molecules, since they appear to remain associated with vesicles after clathrin release both by the uncoating ATPase iiz iGlro(2x.29)(see below) and irz ~ i v o ( ~In~this ). model, phosphorylation or receptor tails by the AP-associated kinase (see below) could be involved in either vesicle targeting or coat dissociation. Finally, since many reactions are reversible in vitro , although unlikely, it catmot be rulcd out that APs may play a role in clathrin disassembly in ITiiw . This possibility finds some support in a recent report that APs may stimulate the uncoating reaction in ~ i f r o ( ~ ’The ). involvement of APs in coalcd pit initiation, invagination and budding has not been measured directly iii vitro. Therefore. it is important to consider these and other models for AP function in vivo.

Coated Vesicle-associated Kinases Several distinct kinase activities have been reported to copurify with coated vesicles. The 5OkD subunit of AP2 is a kinase based on its ability to be autophosphorylated in ~ i t r - 0 ' ~Its ~ )in) . vivo substrate(s) are unknown. Also associated with coated vesicles is a casein-kinase 11-like activity found to phosphorylate LCb in ~ i t r o ( LCb ~ ~ )phosphorylation . is stiinulated by poly-I-lysine and other polyamines. The 4.5 kD catalytic subunit of this kinase is resolved h m the major coat constituents by gel filtration chromatography. A recent report, however, has suggested that this casein kinase activity may correspond to the 47 kD subunit of APl which appears to phosphorylate both LCb and the CI-M6P-receptor iiz vitr-o and perhaps in ~ i v o ( It ~~ was ) . proposcd that this latter activity may be important for regulating API -tail interaclions in v i ~ ~Due n . to apparent differences in gel-filtration and extraction properties, it remains uncertain as to whether the 47 kD subunit does, in fact, corrcspond to the CV-associated casein kinase I1 identified earlier(33)).It is worthwhile noting thal neither the SO kD nor the 47 kD subunits show any sequence homologies with known kinase@) . In addition, it has not been determined whether the S0kD subunit of AP2 also phosphorylates receptor tails. Two additional CV-associated kinases have been reported; an unidentified Ca2+, calmodulin and CAMP-independent kinase which copurifies with API and phosphorylates the SO kD subunit of AP2 and an adaptin kinase apparently not associated with either AP Thus, without unambiguously identifying the polypeptides responsible for these kinase activities, there may be as many as five distinct CV-associated kinases. Physiological roles for these kinases have yet to be demonstraled. The physiological substrates for these kinase activities are unknown. However, many coat constitutcnts arc phosphorylated in vivo including the 100-110 kD and 50 kD subunits of APs? Lcb and, to a lesser extent. LC,(37,38),Although the functional consequences of these phosphorylation events are unknown, quantitative differences were found in the extent of phosphorylation of coat proteins present in assembled vs. unassembled p 0 0 l s ( ~ ~Whether ~. these phosphorylation events are.due to CV-associated kinases or other unidentified kinases, is not known.

A Dynamic Cycle of Coat Assembly and Dissassembly The dynamic cycle of coat assembly and disasscmbly illustrated in Figure 2 is intimately associated with the overall mechanism of receptor-mediated endocytosis . In the following sections, many unanswered questions regarding this pathway will become apparent as each \tep is examined in detail.

Coated pit assembly, growth and invagination Coat proteins appear to assemble onto the cell surface as planar structures which later gain curvature forming invaginated pits(3"). Receptors bearing the appropriate 'endocytosis' signal on their cytoplasmic tails are concentrated into coated

pits. Do receptor tails initiate coat protein assembly or do receptors migrate into coated pits after assembly is initiated? Two studies using transfected cclls expressing high levels (>3 x 106/cell) of human transferrin receptors (Tfn-R) suggest that clathrin assembly on the cell surface is responsive to increased receptor nunibers(40.41).However, when the recruited clathrin lattices were more carefully examined using a surfacc replica technique, Miller et al.'41)lound them to be planar structures whose morphology was distinct from that of coated pits. These authors showed further that the liaction of transferrin receptors found associated with these clathrin lattices decreased at high levels of expression. These results suggest that other factors involved in coated pit assembly, invagination and receptor-sorting might become limiting in cclls expressing abei-rantly high numbers of receptors. If receptor tails can recruit clathrin to the membrane, what prevents clathrin-coated pit assembly onlo receptors in other intracellular locations? For example, the Tfn-K is more highly concentrated o n endosomal membranes than on the cell surface. What other factors, membrane or cytosolic, ensure that coat assembly is initiated only on the appropriate nienibrane? Although clathrin self-assembly and rebinding to membranes in vitro is spontaneous, morphological and biochemical studies using cell-free assays (see below) have suggested that de n o w formation of coated pits on the cell surface is both cytosol and ATP-dependent. These results suggest that other factors might regulate the initiation or nucleation of coat assembly at the cell surface.

Coated vesicle budding Coated vesicle formation involves not only the completion of an enclosed coat struchire. but also a membrane fission event to form a sealed endocytic vesicle. Earlier results had suggested that a single-round of endocytosis could occur in ATP-depleted cells providing support for the notion that clathrin assembly provides a 'motor' which drives membrane invagination and budding("). However, it was recently demonstrated in ATP-depleted cells that although coated pits could efficiently invaginate to sequester ligands from bulky extracellular probes, coated ve cle budding ( i c . membrane fission) was severely inhibited( 1. It is not known how ATP is utilized to drive mcmbranc fission. Recent genetic evidence has demonstrated a role for another class of proteins in coated pit invagination and coated vesicle budding. Drosophila bearing the shibire mutation have a pleiotropic temperature-sensitive defect in endocytosis which results in accumulation of coated pits at thc cell surface'"). The gene responsible for the shibire defect has recently been and found to be 70% identical to rat brain dynamin, a microtubule-stimulated GTPase with microtubule bundling and motor activity(47). This finding demonstrates at least one enzyme required for coated vcsicle forniation which has not been identilied as a coated vesicle constituent. Are there other such factors? What role does dynamin play in coated pit invagination and budding? This question is particularly intriguing since drugs which perturb

1. initiation/ nucleation

2. growth

3. in vagination

+

6

GTP

GDP

ATP \

Y

\

%P

2 l YY

p % @

$a

y d

%P

4.

/

/

budding/ fission

CV-associated kinases? / /

GDP

A

+&+

Y

Y Y

Y

triskelion

Y

i

5 . clathrin release uncoating A TPase?

6. AP release?

AP

- receptor ligand --C

AP binding site ?

Fig. 2. The dynamic cycle of coat aasembly/diassenibly which drives receptor-mediated cndocytosis. Reactions 1 through 7 constitule a model [or thc dynamic cycle of coat assembly and dissassembly which is coupled to receptor-mediated endocytosis. Indicated are some of the, as yet, unanswered issues with regard to the mechanisms which drive this complex overall process.

microtubules in viva have no detectable effect on receptormediated endocytosis.

The uncoating reaction Morphological studies have shown that rapidly after formation of coated vcsicles, the coat is shed. A 70 kD 'uncoating ATPase' which catalyzes the ATP-dependent release of clathrin from empty cages and coated vesicles in vitro has been identified and is identical to the heat shock cognate protein, h s ~ 7 0 1 ~The ~ ) .uncoating ATPase releases clathrin from coated vesicles but not apparently from deeply invaginated coated pits(29).How does the enzyme distinquish between these two structures? Light chains are important for uncoating ATPase recognition of clathrin cages(48)and recent evidence suggests that conformational changes of LCa might be important for regulating the uncoating reaction("). Clathrin is released as a stoichiometric complex with uncoating ATPase. Enzymatically released clathrin will not reassemble to form cages. Are other factors required to restore clathrin's assembly competence andlor to direct clathrin assembly at the cell surface:' Assembly protein complexes are not released by purified uncoating ATPase and may themselves be delivered to endosomes. How are APs recycled?

The unassembled pool of coat proteins A variable, but large cytoplasmic pool of clathrin exists in all cells(50):its cytoplasmic concentration can be estimated to be in the M range. It has been assumed, but not directly measured, that a corresponding pool of soluble APs also exists. These concentrations are well within the ranges observed in vitro for the spontaneous assembly of clathrin and APs into empty coats, for clathrin and AP binding to stripped vesicles and for AP aggregation. What then, prevents these spontaneous and apparenlly futile self-assembly reactions and ensures that assembly is appropriately directed toward the cell surface? Empty coat structures have been observed in ATP-depleted cells(s1), suggesting that maintenance of this unassembled pool is energy-dependent. The distribution of clathrin in assembled and unassembled pools is also responsive to hormonal ~tirnulation'~~)). What other factors regulate clathrin and AP assembly in cells? Cell-free Assays for Coated Fit Assembly and Receptor-mediated Endocytosis Clearly many niechanistic questions remain rcgarding recep-

tor-niediated endocytosis (Fig. 2). The recent development tion was measured indirectly as the disappearance of clathrin of cell-free assays which directly measure coated pit asseinfrom the cell surface. Although nuclcotjde-dependent, bly, invagination, coated vesicle budding and receptor-sortclalhrin ‘disappearance’ was not energy dependcnt: both ing should provide a means for addressing these open issues. ADP and ATPyS (a nonhydrolyzable ATP analogue) could In an important extension of studies on the rebinding of coat substitute for ATP. Clathrin disappearance also required proteins to stripped vesicles, Anderson and c ~ l l e a g u e s ( ~ ~ - ~nonphysiological ’~ levels of Ca2+(maximal loss at 500 pM) have developed an assay system which measures coat asscmand was no1 sensitive to GTPyS (a nonhydrolyzablc GTP bly onto the cytoplasmic surface of plasma membranes. Cells analogue). Thus the physiological significance of the events are plated on poly-I-lysine coated surfaces, disrupted by sonmeasured by this approach are unclear. ication to expose the cytoplasmic surfaces of the attached A second a p p r o a ~ h ( ~provides ~ - ~ ~ )a more functional assay membrancs and extracted to remove endogenous coat profor coated pit assembly, invagination and coated vesicle budteins. Extraction with a low ionic strength pH 9 buffer ding by directly measuring the receptor-mediated endocytoremoves 70-80% of bound clathrin while extraction with sis of transferrin into perforated human A43 1 adcnocarcihigh Tris buffers removes -80% of both clathrin and AP noma cells. Perforated cells are prepared by scraping molecules. The rebinding of coat proteins to these stripped adherent A431 cells so as to rcmove small poitions of their membranes is then measured morphologically or biochemiplasma membrane. As a consequence, the endogenous cally. cylosol can be deplcted and the cytoplasmic surfaces of intraClathrin latticcs reassemble onto pH-stripped membranes cellular membranes can be fully accessed by exogcnously following the addition of either c y t o ~ o l (or ~ ~purified ) coat added cytosolic factorsl antibodies, etc. [125T]Transferrin proteins(”~~~’. Clathrin binding is saturable. indicating that which is biotinylated via a clcavahle disulphide bond (refthere exists a limited number of ‘assembly sites’ on the memered to [”sl]BSST) is used as a marker for internalization. branes. Putative clathrin ‘assembly’ sites which appear as The formation of deeply invaginated pits and coated vesicles patches of clustered particles corresponding to coated pits in is measured by a loss of accessibility of [1251]BSSTto high size and shape have been detected on pH-stripped niemmolecular weight probes such as anti-transferrin antibodbranes by rapid freeze-deep etch techniques. As for binding i e ~ ‘ j ~ , or ~ *avididS9). , The acquisition of resistance lo low to stripped vesicles, clathrin binds to pH-stripped plasma moleciilar weight membrane impermeant reducing agents, membranes with a ten-fold higher affinity (-1 0 nM) than APprovides a direct measure of transferrin internalization into clathrin intcractionsi54). The clathrin lattices fonned are of scaled coated vesiclcs(3”s8.s9). By measuring distinct but defined sizes suggesting some mechanism of controlling the overlapping events in endocytosis, these assays provide a extent of clathrin polymerization. means of biochemically dissecting the complex overall Purified APs will also bind to Tris-stripped m e r n b r a n e ~ ( ~ ~ J process of coated pit assembly. invagination and budding. with somewhat lower affinity (-100 nM). AP-binding is satAn cxamination of the rcquiremcnts for reccptor-mediated urable and appears to be mediated by as yet unidentified inteendocytosis in perforated cells has shown that both 6 novo gral membrane protein(s) based o n proteasc-sensitivi ty and coated pit assembly and coated vesicle budding are temperaresistance to carbonate washing(53’.Saturation occurs at I .5ture-. cytosoland ATP-de~endentc~’.~~). Morphological 2 fold higher than endogenously bound levels of APs sugstudies showed that the invagination of preformed coated gesting either that the binding sites are not fully occupied by pits also required elevated These properties endogenous APs or that some AP self-aggregation may are consistent with requirements for endocytosis in intact occur in vitm. Since both stripping conditions leave a significells and distinguish this assay system from the tempcraturc cant fraction of clathrin and APs (-20%) bound to the memand energy-independent clathrin and AP binding assays brane, it remains uncertain as to whether these residual coat described above. Transferrin internalization in vitro can be proteins can provide nuclealion sites for additional coat inhibited by anti-clathrin antibodies and by antibodies assembly. Interestingly, in this system, clathrin will not bind directed toward the cytoplasmic tail of the transferrin recepto Tris-stripped membranes even after rebinding APs to their tor. The assay is also inhibited by nonhydrolyzablc GTP anaoriginal (or higher) Icvels(j5).As for binding L o stripped vesilogues (Carter, Redelmeier. Woollenweber and Schmid, subcles, the rebinding of clathrin and APs to plasma membranes mitted), confirming a role for at least one GTPase (possibly occurs at 4”C, docs not requirc energy (i.c. ATP hydrolysis) dynamin) in receptor-mediated endocytosis in vitro. In and is independent of other cytosolic factors. addition, preliminary fractionation results suggest that disFuturc htudies using this assay systcm should enhance our tincl. as yet unidentified, cytosolic factors are required for knowledge of AP-clathrin-membrane interactions. However, each stage in endocytosis (Smythe, Carter and Schmid, subsince Lhc reassembly is ncilher temperature-, energy- nor mitted. Given thesc properties, Lhis approach promises to cytosol-dependent, this system is less likely to provide provide a useful fiinctional assay for understanding the role insight into energy-requiring events which might, for examof known coat proteins in the sequestration of receptor-ligple. be required for initiating coated pit assembly, or for and complcxcs and for identifying new factors which might mediating coated pit invagination and coated vesicle forinamediate coated vesicle formation. tton. Our understanding of the mechanisms of receptor sorting An extension of this approach was recently developed to and endocytosis should grow rapidly as the wealth of bioassay for coated vesicle budding from purified plasma m e n chemical information on coat prolcins is applied to these branes adsorbed to poly-l-1ysine(s6). Coated vesicle forma-

new assays for coated pit assembly, invagination and coated vesicle budding.

Acknowledgements I wish to thank Drs. Velia Fowler, Thomas Redelmeier and Elizabeth Smythe for helpful discussions and careful reading of the manuscript. Research in my laboratory has been supported by NIH grants GM42445 and CA27489 and by the Lucille P. Markey Charitable Trust in the form of a Lucille P. Markey Scholarship. References 1 Tram-bridge, IS. (1991). Endocyrosis and signals for internalization. Curr. 0.Cpll Bid. 3,634-64 I. 2 Pearse, B.M.F. and Cruwther, RA. (1987). Structure and assembly OF coated veaiclm. Annu. Rev. Biophys. Biophys. Chem. 16,49-68. 3 Brodsky,F.M. (1988). Living witb clathrin: ikrule in intracellularmembranetr~fic. Science 242,1396-142. 4 Morns, S.A., Ahle, S. and Ungewickd. E. (1989). Clathrin-coated vesicles. Curr. Op. CellUiol. 1,684-690. 5 Parse, B.M.P. and Robinqon, M.S. (1990). Clathrin, adaptors, and sorting. A m . Rev. CellAiol. 6: 151-171. 6 Keen, J.H. (I9!!0). Clathrin and associated assembly and disassembly proteins. Annu. Rev. Bioctiem. 59.41s-438. 7 Brodsky, F.M., HiU,B.L., Acton, S.L., NathkaL, Wong, D.H., Ponnambalam, S. and Parham, P. (1991). Clathrin light chains: arrays of protein motifs that regulate coated-vesicledynamics. TIRS 16,208-213. 8 Schmid, S.L.,Mutsumoto, A. K. and Kothman, J.E. (1982). A domain of clathrii that forms coats. Proc. Xatl A c d . Sci. USA 79,9 1-95 9 Winkler, F.K. and Stnnley, K.K. (1983). Clathrin heavy chain, light chain interaction. € M E 0 J. 2,1393-1400 10 Robinson, M.S. (1 987). IWkD coated vesicle protcins: Molecular heterogeneily and inkacellulardistribution studied with monoclonal antibodies.J.Cell B i d . 104,887895 11 Ponnambalam, S., Robinson, M.S., Jackson, A.P., Peiperl, L. and Parham, P. (1990). Conservationanddiversiry in families of coatedvesicle adaptins. .I. B i d . Chem. 265,4814-4820. 12 Rohinson. MS. (1990). Cloning and expression of y-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus. J. Cell B i d . 111,23192326 13 Kirchhausen, T., Davis, A.C., Frucht, S., O'Brine tirm, B., Payne, G.S. and Tubh, R. (1991). AP17 and AP19, thc mammalian small chains of the clathriiassociated protein complexes show homology to Yapl7p. thcir putativc homolog in yeast.J.Bio1. Chem.266,11153-11157. 14 Heuser,J.E. and Keen, J. H.(1988). Deep-etch visualization of pmteins involved in clathrin assembly. J. CeNBiol. 107,877-886 15 Mnrphy, J., Ileasure, I.T., Puszkh, S. I'rasad, K. and Keen, J.11. (1991). Clathrin assembly protein AP-3: The identity of thc 155K protein. AP 180, and NP185 and demonstration of a clathrin bidning domain. J. H i d . Chcm. 266.4401-4408 16 Ahle, S. and Cingewickell, E. (1990). Auxilin, a newly identified clathrinassociatedprotein in coated vesicles from bovine brain. J. CeNBiol. 111,19-29. 17 Zaremba, S. and Keen, J.H. (1983). Assembly polypcptidcs from coated vesicle8 mcdiate reassembly of unique clathri cwts. J. Cell B i d . 28,47-58 1 R Ahle, S. and Ungewickell, E. (1989). Identification of a cldthrin binding subunit in the HA2 adaptor protein complex. J. B i d . Chem.264,20089-20093. 19 Keen, J.H., Beck, K.A., Kirchhausen, T., and Jarrett, T. (1991). Clatbrin domains involved in rccognition by assembly protein AP2. J. Hwl. Ch!m. 266,79507956. 20 Scbroder, S. and Ungewickell, E. (1991). Subunit intcracrion and function of clathrin-coated vesicle adaptors from the golgi and the plasma mcmbrane. J. Bid. Chem. 266,7910-7918. 21 Prasad, K. and Keen, J.H. (1991). Interaction of assembly protein AP2 and its iwlaledsubuniLswith clathrin. Hiwhem. 30,5590-5597. 22 Hmspul, M., Luna, E:. and Rrantnn, D. (1984). The association of clathrin fragments with coated vesicle membranes. J. B i d . (:hem 259, 1 1075-1 1082. 23 Vishup, D.M. and Bennett, V. (1988). Clathrin-coated vesicle assembly polypeptides: physical propcrties and rcconstitution studies with brain membranes. J. Cell Bid. 106,39-50. 24 Penrsc. B.MB. (I 9881. Receotors comnete rtrr adantors found in nlasma mcmbrane coatedpits. € M E 0 J. I , 3331-3i36. 25 Glickman, J.N., Conibear, E.,and Pearsc, B.M.F. (1989). Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-likegrowthhctor II receptor. EMBO.I.8, 1041-1047. ~~

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26 Chm, D.J., Strauhinger, RM., Actun, S, Nuthke, I. andBrudsky, F.M. (1989). 100-kDa polypeptides in peripheral clathrin-coated vesiclcs are rcquired For receptormediated endocylosis. Proc. Nut1Acud. Sci. USA %. 9289-9293. 27 Beck, K.A. and Keen, J.H. (1991). Self-association of the plasma mcmbraneassociatcd clathrin assemhly proteinAF'2. J. B i d . Chem. 266,4437-4441. 28 Rothmnn, J.15. und Schmid, S.L. (1986). Enzymatic recycling of clatbrin from waled vesicles. Cell 46.5-9. 29 Heuser, J. and Steer, C.J. (1989). Trimeric hinding or the 70-kD uncodting AIPase to the vertices of clathrin triskelia: a candidate intermediate in the vcsicle uncoating rcacti0n.J. Cell Hid. 109,1457-1466. 30 Guagliardi, T..E., Kopplman, B., Blum. J.S., Marks, M.S., Cmswell, P. and Brodsky, FM. (1990). Co-localization of molecules involved in antigcn processing andprcscntation in an early endocytic compartment. Nature343.133-139 31 Bamnch, W., Prasud, K., Greene, L. and Eisenberg, E. ( I W I ) . 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56 Lin, H.C., Moure, M.S., Sanan. D.A. and Anderson. R.C.W. 11991). Reconstitution of clathrin-coated pit budding fioiii plasma membranes. J. Cell B i d . 114.881-S91. 57 Smythe, E., Pypaert, M., Lucocq, J. and Warren, G. ( 1 989). Foination of coated V ~ S I C I Z I frorn coaled pilu in hmken A131 celh J . C’rliBiol. 108, 843-851. 58 Schmid, S.L. and Smythe, E. (1991). Stage-specific assaya for coated pit formation and coatcd vesicle budding in vitro J . Cell Biol. 114,869-880. 59 Smythe, E., T.E. Redelmeier and S.L. Schmid (1992) Rcceptoi-mcdiated

endocytosis in semi-intact cells In Methods in & : w d o ~ s , YOI 219. -(cd. J.E. Rothnian ), pp. 223-231, in press. 7~

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Immunosuppressive and Anti-inflammatory Drugs April 12, to 15, 1993 Orlancio Marriott International, Orlando., F loritla Currcntly used immunosuppressive and anti-inflammatory drugs have limiting toxicitics. Rccently the intracellular ligands of glucocorticoids and immunosuppressive drugs have been defined, and their mode of action has heen elucidated at the molecular biological level. New imrnunosuppressivc drugs have been developed and arc in clinical trials. These include a purine synthesis inhibitor-a pro-drug of mycophenolic acid; a pyrimidine synthebis inhibitor-brequinar. and thc niacrolides, FK 506 and rapamycin. Observations on thcse and other immunosuppressive and anti-inflammatory drugs in cxpcrirnental animals and man will be reviewed at the confcrencc. Major applications to be considercd are prevention of organ graft rejection, improvcd treatment of rheumatoid arthritis and liver disease and the early diagnosis and prevention of insulin-dependent diabetes mellitus. Anthony C. Allison Vice President for Rcsearch Syntex Research 3401 Hillview Avenue Palo Alto, CA 94304

Chference Steering Commitfee: Hans Fliri Assistant Dircctor Preclinical Research Sandoz, Basel CH-4002 Switzerland

Kevin Lafferty Director Barbara Davis Center for Diabetes Kesearch University of Colorado Denver, CO 80262

Therc will bc contributed poster sessions in conjunction with this confcrencc and these will form an integral part of the program. The deadline for submission of poster abstracts is January 7, 1993. The entire abstract, including title, author(s), and affiliations, must be typed single-space and contained within a rectangle that measures 5” X 4r (w x 1). (Abstract form is not necessary.) Abstracts should be sent to Dr. Allison at the abovc address. For furthcr information contact: Conference Dcpartmcnt, New York Academy of Sciences.

The mechanism of receptor-mediated endocytosis: more questions than answers.

Receptor-mediated endocytosis occurs via clathrin-coated pits and is therefore coupled to the dynamic cycle of assembly and disassembly of the coat co...
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