Turnover of the plasma membrane of mammalian cells

Life Sciences, Vol . 24, pp . 951-966

Pergamon PressPrinted in the U.S .A .

MINIREVIEW

TURNOVER OF THE PLASMA MEMBRANE OF MAMMALIAN CELLS

Darrell Doyle and Heinz Baumann

Department of Molecular BiologyRoswell Park Memorial InstituteBuffalo, N.Y . 14263

Our purpose in writing this review is to analyze the variousways by which a protein or a set of proteins might be insertedinto and removed from the plasma membrane of a mammalian cell .We w9.11 consider primarily those membrane proteins whichpenetrate at least into the lipid bilayer, and particularly thosepolypeptides that span the lipid bilayer and are externallydisposed at the surface of the cell . External labeling methods,especially those using either lactoperoxidase-catalyzediodination (1,2) to label covalently available tyrosines or thegalactose oxidase-tritiated NaBH4 reduction method (3-5) to labelsugar moieties allows, in theory, unequivocal identification ofexternally oriented proteins, glycoproteins, and glycolipids ofthe plasma membrane . This means of identifying authentic plasmamembrane constituents is more reliable, we feel, than are cellfractionation techniques followed by chemical and biochemicalanalyses of isolated cell fractions because it is difficult, ifnot impossible, to obtain a homogeneous plasma membrane fractionfrom a tissue culture cell . External labeling methods are notfree of problems ; for example, polypeptides superficiallyadsorbed to the cell will also be labeled . This class of proteinscould complicate membrane analyses, but as we,discuss later, itis possible to distinguish between adsorbed and intrinsic (6)membrane proteins . We consider first the degradation of plasmamembrane proteins because experimentally it is easier to followthe fate of plasma membrane proteins after external labeling ofcells in situ than it is to study the synthesis and insertion ofthese proténs back into the membrane . Further, the mechanismsemployed by the cell to degrade the surface membrane placesrestrictions on the mechanisms that can be used in the biogenesis(7) of the proteins forming this membrane . In Figure 1, isshown all or most of the conceivable modes by which a membraneprotein or a set of membrane proteins could be eliminated fromthe cell surface . For simplicity, we consider mainly tissueculture cells as models which are hopefully representative forother mammalian c~in situ in the animal .

The pathways) of membrane protein (and lipid) degradationinclude : (1) Interiorization of a unit of membrane followed byfusion with a lysosome and degradation of the entire interiorized unit, both protein and lipid (Steps H, N f P, in Figure 1) .(2) Aggregation of specific proteins in the membrane into a"patch" or a "cap" followed by interiorization of the unit of

0024-3205/7/110951-1602 .00/0Copyright (c) 1979 Pergamon Press Ltd

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FIG . 1

Possible Modes of Membrane Biogenesis andTurnover in Animal Cells . A) Biosynthesis of theprotein backbone of plasma membrane proteins ;beginning of glycosylation (N-glycans) . BjGlycosylation (terminal, 0-glycans) continued in Golgior GERL ; budding of plasma membrane precursor vesicle .C) Precursor fuses with the plasma membrane . D)Synthesis of skeletal and contractile elements of themembrane ; association of these proteins with theprecursor vesicle . E) "Shedding" of plasma membraneproteins . F) Cleavage of plasma membrane proteins(proteolysis, glycosidases) . G) t'xpulsion orexocytosis of plasma membrane ("shedding" of wholemembranes, footprints, etc .) . H) lnternalization ofplasma membrane units (including pinocytosis,phagocytosis) . 1) Reinsertion of non-degraded,recycled plasma membrane vesicle . K) Patching byantibody, lectins, etc . L) Lapping (cytoskeletalelements omittedl) . M) lnternalization of cappedmaterial . N) Fusion of internalized or plasmamembrane reservoir vesicle with primary lysosomeforming a secondary lysosome . P) Degradation of thevesicle in the secondary lysosome . R) Recycling ofskeletal elements, or degradation of these elements .

membrane containing the aggregated proteins, and degradation viathe lysosomal pathway (Steps K, L, M, N, $ P, in Figure 1) . (3)Shedding into the medium (or exocytosis) of a unit of plasmamembrane with all of its constituent proteins, sugars and lipids(Step G, in Figure 1) . (4) Shedding (or secretion) of individualpolypeptides from the membrane into the medium (Step E, in

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Figure 1) and (5) Degradation of an externally disposed plasmamembrane protein by a protease or glycosidase, which is itselfa plasma membrane constituent or which has been released fromthe cell into the medium (Step F, in Figure 1) .

Based on thermodynamic and other considerations which arepresented in detail elsewhere (7), we do not consider as likelya mechanism of degradation whereby a transmembrane protein orglycoprotein is able to dissociate from the lipid bilayer andenter the "soluble" cytoplasmic pool where it is subject todegradation (8) . That is, proteins do not as a rule continuallydissociate from and reassociate with the lipid bilayer . However,a polypeptide can be secreted from the cell or released from themembrane (F, E) into the medium and then reassociate again withthe surface of the cell . Similarly, a "membrane associated"cytosolic protein such as actin, myosin, or tubulin or otherproteins forming a "subskeletal" system of the membrane couldbecome dissociated and enter another cell compartment ; but, thisclass of proteins does not penetrate much, if at all, into thelipid bilayer (9, 10) .

Although each of the modes of plasma membrane proteindegradation shown in Figure 1 has been found to be used byvarious mammalian cells, not every cell will use every mode tothe same extent . For example, in suspension cultures ofhepatoma cells, such as rat MH1C1 cells, H-35 cells or HTC cells(11-16) and mouse macrophages (17,18), the primary mode ofdegradation of the plasma membrane is by interiorization of aunit of membrane followed by fusion of this unit with a lysosome .The polypeptide composition of the interiorized unit or domainof plasma membrane can be very complex . For example, there areas many as 100 different polypeptides externally oriented in theplasma membrane of these hepatoma cells . Indeed, thesepolypeptides form the bulk of the membrane protein . Yet, mostof these polypeptides (12), as well as the glycolipids (14) ofthe hepatoma cell plasma membrane turn over at similar rates withfirst-order or pseudo first-order kinetics, i .e . as unit, withhalf-lives of 100 hours or more . Figure 2 illustrates thisslow rate of degradation of surface iodinated proteins in HTCcells . The rate of loss of the incorporated radioactivity fromthe membrane constituents is not dependent on either cell growthbehavior or the extent of iodination . Homogeneous, 3or nearlyhomogeneous rates of degradation are also shown by H-fucose-labeled membrane glycoproteins of i~ilCl cells, as illustratedin Figure 3 .

It is not yet clear whether the membrane associated proteinsthat underlie the lipid bilayer on the cytoplasmic side of thecell are degraded at the same time as the more integral proteinsof the plasma membrane . These proteins, such as actin, myosinor tubulin may be degraded by a different mechanism and atdifferent rates (Steps P, R, in Figure 1) than the transmembraneproteins . Similarly, these peripheral or "subskeletal" typeproteins are probably synthesized via a different mechanism,possibly on free polysomes (Step D) (19) than are the externallyoriented plasma membrane proteins (Steps A, B, C, D, in Figure 1 ;see later discussion of this point) .

Long half-lives (100 hours or more in HTC cells) relative to

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A

FOURS AFTER LABELLING

FIG . 2

Degradation of 125I_labeled Plasma M~mbraneProteins of H'1'C Cells . ~~~ cells (2 x 10 ) wereiodinated with 5 mCi Na

I in the presence (A) orabsence (B) of 6.16 mM Na 125 1 as describedelsewhere (11, 12) . The cells were washed threetimes with Earle's balanced salt solution and twicewith growth medium . The cells were suspended ingrowth medium ~t the cell densities indicated andcultured at 37 C . The medium was replaced inexperiment A completely after 72 hr ., and inexperiment B the culture was diluted with freshmedium after 72 hr . and 118 hr . At the timesindicated, aliguots of 10 ml of cell suspensionwere removed, the cell density was determined andthe cells were collected by centrifugation . Thecells were washed three times with Earle's balancedsalt solution . The cells were precipitated with10~ trichloroacetic acid and the lipid extractedtwice from the pellet with chloroform/methanol(Z :1) . The soluble, as well as insoluble fractionswere solubilized and counted in a liquidscintillation counter . The culture medium wascentrifuged for 30 min at 200,000 g . Totalradioactivity as well as that in acid and ethanol-insoluble fraction (medium protein) was determined .All values for the radioactivity were normalized tolU ml of original cell suspension and represent theaverages of the duplicate determinations .

Vol . 24, No . 11, 1979 Mammalian Cells Plasma Membrane Turnover

FIG . 3

Degradation of 3H-fuco-se-labeled Membrane Gly-coproteins of MH 1C1 RatHepatoma Cells . MH1 C1cells growing in mono-layer (25 cm2 ~ werelabeled with H-fucose(18 .5 Ci/mmol, 40 uCi/ml) for 24 hr . Themedium was removed andthe cells washed threetimes with unlabeledmedium . The cells werethen cultured for anadditional 3 days inmedium containing 0 .1mM L-fucose . Themedium was changeddaily . Immediatelyafter the pulse (a)and 24 hr (b), 48 hr(c) , and 72 hr ~d)after chase, the cellsof one flask werescraped from thesupport and washedthree times withphosphate bufferedsaline . The cellswere sonicated in 50mM Tris HC1 pH 7 .8 con-taining 1 mM PMSF andthe homogenate cent-rifuged at 200,000 gfor 60 min . The pellet(membrane fraction) waswashed once and thendissolved in SDS-samplebuffer . Aliguotscontaining 40,000- .70,000 acid insolublecpm were electrophoresedon a 28 cm long SDS-polyacrylamide gel witha linear gradient ofacrylamide from 6 .5to 12~k . The fluor-ogram of the gel aftera 2-week exposure isshown .

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the doubling time of the cell (24-30 hours in HTC cells) seem tobe characteristic of the endocytotic mode of plasma membranedegradation not only for hepatoma cells but also for a varietyof other mammalian cell types (18, 2U, 21) . Actually theturnover of these externally disposed membrane proteins is morecomplicated than just suggested in that they often show biphasicturnover after external labeling either by lactoperoxidase-catalyzed iodination of cells or by tritiated borohydridereduction of oxidized galactose residues of cell membraneglycoproteins . That is, after labeling, the membrane proteinsfirst turn over at rapid rates (half-lives of 1 day or less)than at slower rates (half-lives of days) (12, 14, 18) . For themost part, the same set of membrane proteins turn over at the twodifferent rates . While part of the more rapid phase of turnovercould be due to a response of the cells to the handling duringlabeling, it is also possible that two or more differentpopulations of cells or membrane (in terms of function, or stageof the cell cycle) exist in the culture . Much more work isrequired to distinguish among these possibilities .

Unit turnover suggests that the l0U or so externallyoriented polypeptides are distributed more or less at random inthe plasma membrane of the HTC cell . If this is so, then toremove specifically a population of homologous polypeptidesfrom the cell surface by the endocytotic pathway, these moleculeswould have to be first concentrated in the unit of membrane thatis to be interiorized . Indeed, in many diverse mammalian cellsboth divalent antibodies to specific surface proteins and plantlectins, particularly Concanavalin A, do appear to bring about aredistribution, aggregation, and concentration of the specificmembrane antigen or lectin receptors) into regions which havebeen termed patches or caps (22-26) . Mechanisms involvinginteractions of the externally oriented plasma membrane proteinwith intracellular proteins underlying the lipid bilayer of themembrane have been proposed to explain on a molecular basis thisaggregation-disaggregation of membrane proteins (27-32) .Aggregation followed by interiorization (and degradation?) ofexternally oriented membrane polypeptides may also be involvedin many different receptor-mediated interactions of the cell .For example, regulation of the concentration of surface receptorsfor a hormone such as insulin (33-36), or for even largerpolypeptides such as the receptor for low density lipoproteins(37-39) as well as the interaction of various polypeptide growthfactors (40-43) may well include aggregation and interiorizationas a first step in the mechanism of action . If this is trice, byanalogy to the reorganization of membrane proteins brought aboutby antibodies, a receptor protein like that for insulin shouldhave multiple binding sites for the molecule with which itinteracts . While, at least two binding sites would be requiredby an effector molecule, like insulin, to cause aggregation ofthe receptor into a patch or a cap .

Single domain turnover of most of the polypeptides of theplasma membrane, as shown by HTC cells, for example, may besomewhat of an anomaly of the tissue culture system . 'that is,this mode of turnover may reflect a type of structural ormorphological homogeneity of the plasma membrane in these tissueculture cells . For example, suspension cultures of HTC cells donot show the tight or gap junctions characteristic of

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hepatocytes in situ in liver . Perhaps they also have lost otherless obviousiegiônal specializations of the plasma membranes ofhepatocytes and other types of cells in other tissues . It isconceivable and indeed most probable then that other cells,including hepatocytes in the liver, may have more than onedomain in terms of membrane degradation and that differentpolypeptides are restricted tq different domains by constraints(cytoskeletal?) on lateral mobility in the lipid bilayer (44-46) .Hence, rates of total plasma membrane protein turnover indifferent tissues of the animal would be heterogeneous, but thepolypeptides comprising a domain could still all be interiorizedand degraded as units by the endocytotic route .

An important question to consider is whether degradation viathe lysosomal route is obligately coupled to interiorization ofthe unit of plasma membrane . That is : Is every interiorizedunit of plasma membrane degraded? The answer is probably no ;Steinman et . al ., (47) have shown by an electron microscopicstereological analysis of mouse L cells and macrophages pulsedwith horseradish peroxidase that the rate of interiorization ofmembrane is much faster than is the rate of degradation . Asmuch as one-half of the membrane was interiorized in a timeinterval of about one hour while it required 24 hours or moreto degrade one-half of the membrane protein . Since the surfacearea of the cell did not decrease nor did the amount of theintracellular membrane structures containing the pinocytosedhorseradish peroxidase increase, Steinman et . al ., concludedthat the interiorized membrane units were wingrecycled to thesurface (H, N, $ I of Figure 1) . Surprisingly, it appeared thatall of the interiorized units of membrane containing thehorseradish peroxidase did fuse with primary lysosomes . Thehorseradish peroxidase appeared to be degraded while the membraneproteins were spared, possibly because they are intrinsicallymore resistant to degradation and can in some way be pinched offfrom the secondary lysosomes prior to being recycled to thesurface . It is also possible that the plasma membrane proteinsof the secondary lysosomes were recycled through the golgi orGERL (Golgi, Endoplasmic Reticulum, Lysosome) apparatus of thecell (48, 49) . Tulkens et . al ., (50) in a follow up to theSteinman et . al ., study wéreâble to "trap" the recycling membraneunits by incorporating into the lysosomal compartment anantibody made to a primary antibody directed against cell surfaceproteins . The anti-antibody bound to the antibody to the surfaceproteins after the interiorized membrane fused with a lysosome .This presumably prevented pinching off and recycling of theinteriorized plasma membrane .

While the amount of membrane material that is interiorizedper unit time is enormous, the function of interiorization,lysosomal fusion, and recycling of the interiorized membraneunits is not known . However, recycling has the potential ofpermitting the cell to continually monitor (or "taste") theenvironment . A recycling mechanism that functions as part of aninformation gathering and âdaptive response system o-f the cellis an, intriguing but only speculative thought at the presenttime .

In suspension cultures of HTC cells, there is very littleshedding or exocytosis (Step G of Figure 1) of units of plasma

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membrane . Hence, very little large molecular weight materialoriginating from the plasma membrane is found in the growthmedium of these cells . However, the amount of membrane materialshed can be very significant in some other mammalian cells,particularly lymphocytes (51, 52) . Indeed, the main pathwayused by lymphocytes to rid themselves of specific surfaceantigens in response to a challenge with antibody may be byshedding, particularly after the antigen has become concentratedinto a patch or a cap (53, 54) .

Monolayer cultures of fibroblasts also may depositconsiderable amounts of membrane associated material, bothprotein and lipid, on the surface of the support on which theyare growing (55) or moving (56) . In some cells this materialmay arise by membrane shedding while in others large molecularweight proteins are secreted to form an adhesive substratum forthe cell . The LETS, large external transformation sensitive,protein (57-59) which is similar or identical to fibronectin(60), galactoprotein (61), and CSP (cell surface protein) (62)and which may be involved in promoting cell adhesion and cell-cell aggregations among other functions most likely arises by thesecretioy route (63-66) . This surface membrane associatedprotein is currently receiving considerable attention becauseit is reduced in amount in many transformed and malignantcells (67, 68) . lts apparent mechanism of formation, bysecretion followed by absorption back to the cell surface(via a specific externally oriented integral membrane receptorprotein?), may be representative of a whole set of yetunidentified proteins forming a surface coat on many differenttypes of cells (69, 70) . This class of proteins would all belabeled by external labeling methods, yet they are not integralmembrane proteins in that they do not penetrate the lipidbilayer . They could be distinguished from those proteins that dopenetrate or span the bilayer by either tryptic fingerprintcomparison of the protein labeled in situ or labeled afterisolation and purification or by la~e~ of inside-out plasmamembrane isolated on phagocytosed polystyrene latex beads(17, 18, 71) or isolated by wrapping the plasma membrane arounda positively charged bead (72, 73) .

Another way by which some externally oriented polypeptidesare removed from the surface of HTC cells is by cleavage of partof the protruding polypeptide, presumably by a protease(s) whichitself is a plasma membrane protein or which is secreted fromthe HTC cells (14, 74) (Step F, in Figure 1) . Only a few,perhaps only one major HTC cell plasma membrane protein, isdegraded by this mechanism . A 55,000 molecular weightglycopeptide fragment is released from an 85,000 molecular weightplasma membrane glycoprotein at a rate faster than that ofoverall plasma membrane protein turnover when HTC cells aremaintained in serum-free medium . 'l'he parent membrane glyco-protein of this fragment is also very sensitive in situ totrypsin .

The same or a glycopeptide very similarin compositionto that released normally into the medium is also released fromthe cell surface by trypsin . It may be significant that HTC cellsbehave in culture as single cell suspensions but aggregateimmediately after treatment with a concentration of trypsin thatcleaves only (or mainly) the 55,000 molecular weight fragment

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from the plasma membrane glycoprotein . Hence, it is conceivablethat membrane proteases similar to trypsin are involved in cell-cell aggregation by exposing "sticky" ends on the glycoproteinsof two cells . A similar mechanism has been postulated for theaggregation of developing chick cells by Bdelmen (29) .

Although some plasma membrane proteins of HTC cells areremoved by surface proteases (F in Figure 1) the main pathwayof membrane degradation in this cell and many others is asmentioned, the endocytotic one . When the cell in steady stateloses a unit of membrane through this degradatioe pathway, itmust replace at the surface a unit of like composition . One waythat the cell might accomplish the insertion of a complex set ofpolypeptides into the plasma membrane is to have available analready synthesized and assembled unit of membrane in anintracellular compartment . We ~(13) searched for a preformedpool of potential plasma membrane precursor in HTC cells usingfucose as a label for membrane glycoproteins . In HTC cellsthere are fucose-containing glycoproteins which are externallyoriented on the cell in that they can also be labeled bylactoperoxidase-catalyzed iodination and by tritiated NaBH4reduction of cells that had first been treated in situ withneuraminidase and galactose oxidase . When cellswere pulsedfor two hours with labeled fucose, radioactivity appeared in aset of fucose-containing glycoproteins that were present in anintracellular membrane system of the cell . During subsequentculture of the cells in medium containing unlabeled fucose, theset or unit of labeled membrane-bound glycoproteins moved fromthe intracellular compartment to the plasma membrane requiringa transit time of approximately three hours after fucosylationto become externally oriented . However, not all of the fucose-labeled glycoproteins moved from the intracellular compartmentto the plasma membrane . About 60$ of the incorporated fucoseremained as a stable component of the intracellular membranecompartment . Surprisingly, the glycoproteins that remained inthe intracellular membrane compartment were the same as thosethat became inserted into the plasma membrane (Figure 4) . Thisset of membrane glycoproteins was complex when analyzed byelectrophoresis in a highly resolving two-dimensionalacrylamide gel system (75) . Yet each member of the glycoproteinset seemed to be present in the intracellular membrane and inthe plasma membrane in the same relative proportion to eachother . Only one of the total fucose-containing glycoproteinsin the intracellular membrane system seemed to be absent at thesurface . Since these results were unexpected, we wished toconfirm the dual localization of these membrane glycoproteinsby an independent method not based on cell fractionation . We(75) reasoned that if the same fucose-labeled glycoproteins wereboth externally oriented on the plasma membrane and present onan inside membrane of the cell, then it should be possible todifferentiate between these two membrane compartments using aprobe that would in some way react with and alter theglycoproteins of one compartment - the plasma membrane - andnot affect those of the interior membrane system . HTC cellshave a surface glycoprotein with galactose residues accessibleto labeling in situ via the galactose oxidase tritiatedborohydride procure . 'l'his externally labeled glycoproteinwith a molecular weight of 85,000 is, as mentioned above,

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FIG . 4

Glycoprotein Composition of Different MembraneFractions from HTC Cells . ~TC cells were labeledeither enzymatically by NaB H4 reduction ofneuraminidase and galactose ~xidase-treated cells(14), or metabolically with H-fucose (4 uCi/ml ofmedium for 3 days) . 'l'he plasma membrane andintracellular membrane fractions ("microsomal")of the 3H-fucose-labeled cells were isolated by asucrose gradient centrifugation method (13) . Theproteins of the whole cells or cell fractions wereseparated by one-dimensional electrophorèsis on7 .5~ SDS polyacrylamide gels . BPB represent thetracking dye bromophenol blue . jl) Surface-labeled cells (25,000 cpm) (2) H-fucose labeledcel~s (35,000 cpm) (3)-(6) Subcellular fractionsof H-fucose-labeled cells (3) Plasma membranesbanding at the 1 .6 M/1 .8 M sucrose interface(25,000 cpm) (4) Intracellular membranes "lightmicrosomal fraction 1" banding at the 0 .8 M/1 .2 Msucrose interface (30,000 cpm) (5) Intracellularmembranes "light microsomal fraction 2" bandingat the 1 .2 M/1 .4 M sucrose interface (28,000 cpm)(6) Intracellular membranes "heavy microsomalfraction" banding at the 1 .4 M/1 .6 M sucroseinterface (Z8,000 cpm)

completely sensitive in situ to low concentrations of trypsin(14, 74) . However, whén~ls were labeled metabolically withfucose, 70$ of the incorporated radioactivity remained in the

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85,000 molecular weight protein after treatment of the ellswith trypsin . Similarly, sutoradiographic analysis of H-fucose-labeled cells by electron microscopy confirmed thisdistribution of fucose-labeled proteins in two differentmembrane systems of the HTC cell (76) . One of these compart-ments is sensitive to external enzyme probes and one is not .Most of these fucose-containing glycoproteins of the HTC cellalso have sialic acid as the terminal sugar residue . Indeed,much of the charge heterogeneity shown by membrane glycoproteinsin electrofocusing gels is due to these terminal sialic acidresidues . The sialic acid residues also should be accessibleto enzyme modification with neuraminidase if the glycoproteinsare externally oriented on the plasma membrane . Conversely,these residues should be inaccessible if the glycoproteins arepresent inside the cell .

Fucose-labeled HTC cell glycoproteins were isolated byConcanavalin A chromatography . When these proteins were treatedwith neuraminidase, we could show that their electrophoreticmobilities in a two-dimensional acrylamide gel system wereshifted toward a more basic isoelectric point (75j . When thesefucose-labeled glycoproteins were isolated from cells whichwere first treated in situ with neuraminidase, the same glyco-proteins showed thesame shifts in isoelectric point .

Now,however, only a part (3U-40$) of the glycoprotein moved to anew region of the isoelectric focusing gel . Part (60-70$)remained at its original isoelectric point . Hence, only aportion of the fucose-labeled glycoprotein - that on the plasmamembrane - was accessible to the neuraminidase . Most of thefucose-labeled glycoprotein was inside of the cell . Thisinaccessible component could be made accessible to theneuraminidase by breaking the plasma membrane, thereby exposingthe sialic acids of glycoproteins in the intracellular membranesystem . When this was done, all of the glycoprotein componentwas shifted to the new isoelectric point .

The presence of plasma membrane glycoproteins in an intra-cellular membrane system is probably not unique to HTC cells .The acetylcholine receptor of muscle cells, as assayed bybungarotoxin binding, is present on the plasma membrane and aspart of an intracellular membrane system (77, 78) as is thehepatocyte asialoglycoprotein binding protein of liver (79) .Kawai and Spiro (80) have also shown recently that a majorglycoprotein (s) is shared by the plasma membrane and intra-cellular membranes of fat cells . The insulin and growth hormonereceptors may also be present on membranes other than the plasmamembrane (81, 82) .

The large size of the intracellular membrane compartmenthomologous in glycoprotein composition to the plasma membrane,however, may be peculiar to the HTC cell . The function of thislarge reservoir of membrane-bound material identical to theplasma membrane is not known . Our working hypothesis, which weare currently testing, is that in certain periods of its life, acell, not in the controlled environment of a tissue culture, butin the animal, may have to replace a large amount of membrane ina short interval of time . This conceivably could happen atcertain stages of the cell cycle, or perhaps after extensivephagocytosis or as an endocytotic adaptation to a change in the

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environment (patching or capping of a large amount of one or moremembrane polypeptides) . In these cases, the cell might replacethe lost membrane from the reservoir rather than via de novosynthesis .

During the course of biogenesis of membrane proteins (StepsA, B, C and D, in Figure 1) we presume that externally orientedplasma membrane proteins are made primarily on membrane-boundpolysomes in a manner similar to that proposed for the synthesisof secretory proteins (83, 84, 85) . lndeed, it is possible thatexternally oriented or transmembrane proteins differ fromsecretory proteins only in their relative affinities for thelipid bilayer, or in the way in which their "signal" aminoacid sequences are arranged and processed . While there isevidence suggesting that this processing of "signal" sequencestakes place on membranes of the endoplasmic reticulum (86),there is also recent evidence (87) suggesting that secretoryvesicles may fuse with primary lysosomes and that lysosomalenzymes are responsible for processing of secretory proteins .If lysosome-secretory vesicle fusion does indeed occur as astep in secretory protein processing, it might also occur asa step in the biogenesis of the plasma membrane . Hence, it ispossible that the lysosomes could play a role in membranebiogenesis as well as in membrane recycling (see Figure 1), butmuch more work is required to put this hypothesis on a firmexperimental basis .

In summary, we believe that a main pathway for the turnoverof the plasma membrane of most animal cells involves as a firststep interiorization of a relatively large, in molecular terms,unit of membrane . This unit, containing lipids, glycolipids,glycoproteins and proteins is usually not degraded but isrecycled to the surface . When a degradation event does occur,the entire unit is degraded, probably by lysosomal hydrolases,to small molecular weight material . Superimposed upon thisendocytotic pathway of turnover are the other pathways shownin Figure 1 . When a unit of membrane is degraded afterinteriorization, it is replaced by a unit of like compositionwhich has been synthesized and assembled in the intracellularcompartment . We also propose that most cells have some finiteintracellular reservoir of such preassembled units identical incomposition to regions of the plasma membrane . The function ofthis reservoir of potential plasma membrane components is notknown . However, if this reservoir does exist and is common tomany different cell types, it seems reasonable to presume thatit has some function in plasma membrane biogenesis and assembly .For example, little is known about the way the cell places onlyone or a limited subset of proteins into the plasma membrane .This type of replacement of plasma membrane proteins) mightoccur when the concentration of one or a few proteins ismodulated by a hormone like insulin (33-36), or serum proteins(37-39), or specific antibodies directed against surfaceantigens (25, 26) . The cell might increase the concentrationof a protein or a limited subset of proteins in the plasmamembrane by the synthesis and assembly of a new unit ordifferent units enriched in one or more of those proteins to bereplaced in the plasma membrane (Steps A, B, C, D, in Figure 1) .Another mechanism of replacement, however, might be to use the

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already synthesized and assembled reservoir of membrane . In thiscase, new specific proteins might be added to the preassembledunit .

Or, proteins already existing in . the preassembled unitcould be reorganized and concentrated, perhaps by removingrestrictions on lateral mobility . The reorganized membraneunit might then be used as a vehicle to put specific proteinsin the plasma membrane .

fortheH .BFoundation .

Work reported herein from the authors' laboratory issupported by Grants CA 17149, GM 24147 and GD1 19521 .

2 .

3 .4 .

5 .

6 .7 .

ö .9 .

10 .

11 .

12 .13 .

14 .

15 .

Acknowledgment

We thank our colleagues, E . Hou, E . Freedman and J . Twetoperforming some of the experiments and participating indiscussions which lead to the writing of this review .was a postdoctoral fellow of the Swiss National Science

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Documents

Turnover of the plasma membrane of mammalian cells