Sorting of plasma membrane proteins in epithelial cells

Sorting of plasma membrane Morgane

proteins

in epithelial

cells

Bomsel and Keith Mostov

CNRS, Paris, France, and University

of California,

San Francisco,

California,

USA

Proteins follow two routes to reach the correct surface (apical or basolateral) of a polarized epithelial cell: direct sorting from the trans-Golgi network and transcytosis from early endosomes. Several signals have been identified recently that control these sorting events, namely a glycosyl-phosphatidylinositol anchor for apical targeting a 1Gresidue cytoplasmic segment of the polymeric immunoglobulin receptor for basolateral targeting, and phosphorylation of a Ser residue for transcytosis of this receptor. The machinery involved is still poorly understood. Current

Opinion

in Cell Biology

Introduction Asymmetry is an essential feature of most cells in tissues. The cell surface is divided into distinct plasma membrane domains, which serve different functions. The prototype of this asymmetry is exemplified in epitheliai cells, which cover body cavities. They carry out specialized functions in secretion, absorption, and ion transport, and act as a fence forming the interface between the organism and the outside world [ 1,2]. To perform their vectorial functions, epithelial cells have evolved a plasma membrane divided by tight junctions into apical (or luminal) and basolatetal domains that differ in protein and lipid compositions. Despite extensive membrane traffic between these surfaces and intracellular organelles, the polarity of the cell is accurately maintained. A continuous sorting of membrane components has to take place, for which the cell uses several sorting mechanisms.

Pathways

to the cell surface

Newly made membrane proteins destined for both cell surfaces travel together from the rough endoplasmic reticulum through the Golgi and transGolgi network (TGN) [3**]. Delivety of basolateral membrane components is directly from the TGN to that surface in all epithelial cell types examined (Fig. 1). In contrast, delivery to the apical surface vanes according to the cell type. In some cells, apically destined molecules are sorted in the TGN into vesicles that travel directly to the apical surface. Other cells rely on an indirect pathway involving basolateral surface delivery followed by transcytosis to the apical surface, as described below. Once reaching a surface, some molecules form a very stable association with the

CAM--cell

1991, 3647-653

submembranous cytoskeleton [ 4*,5*], with homologous molecules from adjacent cells, or with basement membrane molecules. This selective stabilization may prevent further movement and rapid turnover of these molecules, thereby contributing to the maintenance of plasma membrane polarity. Other molecules are rapidly internalized. Material endocytosed from the apical or basolateml surfaces enters separate apical and basolateral endosomes, which are spatially and functionally distinct [ 6,7**,8]. Approximately 50% of the cell surface is internalized per hour, a flux roughly 10 times that through the TGN. Early endosomes are a major sorting compartment from where molecules are directed to one of three pathways: recycling to the surface of origin; delivery to a prelysosomal compartment (which receives input from both apical and basolateral early endosomes) and transport ultimately to lysosomes; and transport by vesicular tragic to the opposite surface, in a process known as transcytosis, which can occur in both basolateral-to-apicaJ and apical-to-basolateral directions [PJO]. Transcytosis is unique: it the only pathway for polarized sorting common to all cell types examined and in many cases is the principal or sole pathway. MadinDarby canine kidney (MDCK) cells, which form a tight, well polarized monolayer when grown on porous supports in culture, have been extensively used to study polarized sorting [2]. Most plasma membrane proteins in MDCK cells are directly targeted from the TGN to their hnal plasma membrane destination [3,11*,12*], although some proteins reach their ultimate location by transcytosis [9*,13]. In contrast, hepatocytes have no direct TGNto-apical pathway. Instead, all apically destined proteins must first be delivered to the basolateral surface and are then transcytosed to the apical cell surface [ 141. The CaCo2 intestinal cell line exhibits an intermediate phe-

Abbreviations adhesion molecule; CPl-glycosyl-phosphatidylinositol; MDCK-Madin-Darby p@lt--polymeric immunoglobulin receptor; TCN--tram-Golgi network. @ Current

Biology

Ud MN

0955474

canine kidney;

647

648

Membranes Signals Apical

surface

Sorting

for sorting by default

In non-polarized cells, it appears that secretory and membrane proteins do not need any sorting signals to reach the cell surface [22]. Rather they are carried by default in the bulk flow of membrane and enclosed fluid flowing through the secretory pathway. In polarized cells, two arguments favor the proposition that basolateral targeting can occur by default [3**]. First, the TGN-to-apical pathway is absent in hepatocytes. Second, many housekeeping proteins expressed in non-polarized cells are located at the basolateral surface of epithelial cells, while the apical surface contains components that specify the epithelial phenotype. However, to prove that a given marker has no sorting information and is truly carried by default is difficult, especially for membrane markers, as even lipids are sorted 1231.

w

Transcytosis

-9

Basolateral early endosome

Recycling

Basolateral

surface

Fig. 1. Sorting pathways in epithelial cells. Both the vectorial transport and the barrier function of epithelia rely on the polarized distribution of membrane components between the outward-facing apical domain and the inward-facing basolateral domain. Establishment and maintenance of polarity involves two major sites of sorting: the trans-Colgi network CTCN) and the basolateral early endosome. Their relative importance in sorting varies with the epithelial cell type examined as illustrated by the following three model systems. In Madin-Darby canine kidney cells, newly synthesized proteins are primarily sorted in the TGN; direct delivery to either surface occurs from this organelle. In hepatocytes, direct apical delivery from the TCN does not exist. Instead, these proteins must take an indirect route. All proteins are sent to the basolateral surface, and apically destined molecules are endocytosed, sorted in the basolateral early endosome into the transcytotic pathway, and delivered to the apical surface. CaCo2 cells use both the direct and indirect pathways. Delivery to the apical surface, either directly from the TCN or indirectly by transcytosis requires an intact microtubular network, whereas basolateral delivery is independent of microtubules.

notype [ 15-17*,18*]: apical proteins use both pathways to reach the apical surface. In addition to its role in membrane protein sorting, transcytosis also serves one physiologically relevant, epithelial-specific function: the vectorial transport of macromolecules across the cell. Two examples are the basolateral-to-apical transport of polymeric IgA and IgM by the polymeric immunoglobulin receptor (p&R) [10,19*] in mucosal epithelia (Fig. 2>, and the apical-tobasolateral transport of IgG across newborn rat intestines and human placenta by the Fc receptor [20,21].

Signal-mediating

sorting

When a sorting decision must be made it is generally assumed that the molecule possesses a structural recognition motif or sorting signal that contains the necessary information [24]. This signal may positively divert a molecule into a pathway (such as the mannose-6. phosphate signal for lysosomal enzymes, or a conformational motif in the protein sequence, such as a tightturn containing aromatic residues for endocytosis [ 25*‘] > or cause retention in a compartment [for example, the Lys-AspGlu-Ieu (KDEL) signal for endoplasmic reticulum retention]. Early work used mutagenesis to attempt to find characteristic apical and/or basolateral sorting signals in viral glycoproteins [l]. No sorting signals were clearly identified, in part because mutations often disrupted tertiary structure and the oligomerization of these proteins. As discussed below, it is now clear that sorting to both surfaces of the cell can be signal mediated, even if no rules have yet emerged. However, we cannot exclude the possibility that default pathways exist to one or both surfaces.

Apical

signals

The apical surface contains many specialized proteins, and its exoplasmic leaflet is almost entirely composed of glycosphingolipids. Currently, the best defined signal for apical targeting is the glycosyl-phosphatidylinositol (GPI) anchor which attaches certain proteins to the membrane. This class of proteins is found exclusively in the apical surface of most epithelial cells [ 120,261. If the transmembrane peptide anchor of a basolateral protein is replaced with a GPI anchor it is converted into an apical protein [ 12*,27]. The GPI-anchor extends only into the exoplas mic leaflet of the membrane. How can it act as a signal? It has been suggested that in the TGN glycolipids and GPIanchored proteins cluster together by hydrogen bonding in the bilayer, mimicking a microdomain of the apical membrane [3*-l. A sorting protein would recognize these clusters and segregate them into apically targeted

Sorting

of plasma

membrane

proteins

in epithelial

cells Bomsel

and Mostov

vesicles [ 12*]. Clearly, other unknown signal(s) must also exist.

ficient for basolateral targeting. Fusing this segment to a normally apical protein redirects it to the basolateral surface [28].

Basolateral

Recent evidence suggests that the signals for endocytosis [25**] and basolateral targeting are related. Rapidly endocytosed forms of the FcRII IgG receptor, influenza hemagglutinin, and nerve growth factor receptor are all basolaterally targeted, whereas slowly endocytosed forms have an apical distribution (M Roth, personal communication; E Rodriguez-Boulan, personal communication) [29]. However, this correlation cannot be generalized. Mutating the two lyr residues that are part of the internalization signal of the p&R blocks endocytosis, but not basolateral targeting (C Okamoto and K Mostov, unpublished data), while deletion of the pIgR basolateral targeting signal eliminates basolateral targeting but not endocytosis [28]. Moreover, in transgenic mice, the overexpressed human low-density lipoprotein receptor is located basolaterally and not endocytosed in hepatocytes, but is apical and rapidly endoqtosed in kidney tubules [30-l. This result also underscores the importance of considering the function of molecules in various tissues in animals and not just a few cell lines.

signals

If delivery to the basolateral surface were by default, no signal would be necessary. Recently, however, several signals for basolateral delivery have been identified. The clearest example of such a basolateral targeting signal is provided by the p&R, which is targeted directly from the TGN to the basolateral surface (Fig. 2). Mutational analysis has shown that a 17’~amino-acid segment of its 103amino-acid cytoplasmic domain is necessary and suf-

(a)

Apical

surface

SC \

Fig. 2. The polymeric

Basolateral fb)

surface

Extracellular

Intracellular Membrane

Vr Basolateral targeting

:

Avoid degradation I 103-amino-acid cytoplasmic tail

I Liga;fmb;yring l-l

I ! Rapid endocytosis

I

immunoglobulin receptor fplgR): an example of a transcytosed protein. The plgR has been an extremely useful model system for studying polarized membrane traffic. In both Madin-Darby canine kidney cells and hepatocytes it follows the indirect, transcytotic pathway to reach the apical surface and uses both the TCN and the basolateral early endosome sorting machinery. The sorting signals for each step of the cellular pathway of the plgR have been extensively dissected at the molecular level. (a) Cellular pathway of the plgR. The plgR is delivered from the tram-Colgi network CTCN) to the basolateral surface, where it can bind the polymeric immunoglobulin ligand fplg). The receptor and ligand are endocytosed and delivered to the basolateral early endosome. Some receptors are phosphorylated on a particular Ser on its cytoplasmic domain. Phosphorylation probably occurs at the basolateral surface, although this is not well established. In the early endosome, receptors that are not phosphorylated are recycled to the basolateral surface. Receptors that are phosphorylated, or contain an Asp in place of the phosphorylated Ser, are sorted into transcytotic vesicles. These vesicles are delivered to the apical surface. A leupeptin-sensitive protease cleaves off the extracellular portion of plgR. This portion of the plgR is called secretory component (SC) and is released together with the plg into external secretions. fb) Molecular structure and sorting signals of the plgR. The extracellular amino acid terminal portion of the plgR consists of five disulfide-linked domains (I, II, Ill, IV, VI, which are members of the immunoglobulin superfamily. The cytoplasmic, carboxy-terminal domain has been shown by mutational analysis to contain at least four sorting signals. fi) Deletion of the carboxy-terminal 30 amino acids (which comprise a single exon) or mutation of a Tyr in this segment, reduces the rate of endocytosis of the plgR from the basolateral surface by 60%, but has no other effect on plgR sorting. A second Tyr located closer to the membrane is also important for rapid endocytosis. (ii) Deletion of 38 residues from the middle of the cytoplasmic domain yields a receptor that is delivered to the basolateral surface, binds ligand, and is endocytosed, all with near wild-type efficiency. After endocytosis, most of the plgR is directed to lysosomes. This segment of the cytoplasmic domain is somehow involved in avoiding degradation. (iii) The 17 amino acids closest to the membrane are an autonomous basolateral targeting signal. (iv) Phosphorylation of a Ser (located in this 17-residue segment) is the signal for transcytosis.

649

650

Membranes

Transcytotic signals

.

Sorting into transcytotic vesicles occurs in the early endosome. The only known signal for transcytosis is phosphorylation of a particular Ser in the cytoplasmic domain of the pIgR [31**] (Fig. 2). Replacing this Ser with Ala (which cannot be phosphorylated) prevents tmnscytosis, while replacement with Asp (whose negative charge can mimic that of a phosphate group) produces a receptor that is tmnscytosed more rapidly than the wild-type. Phosphorylation cannot be a universal signal for transcytosis, as many transcytosed molecules do not have potential sites for phosphorylation in their cytoplasmic domains. These molecules may use a mechanism analogous to the negative charge of the Asp mutant. Phosphorylation may be a means of regulating the transcytosis of the pIgR.

Machinery

for sorting

We know little about the machinery that recognizes sorting signals and targets proteins to the correct location. The fmt steps in this process are segregation of the molecule to be sorted in the plane of the membrane and formation of transport vesicles. This process is best understood for clathrin-coated endocytic vesicles, where adapter proteins are involved. Adaptins are thought to function either by recognition of endocytic signals on endocytosed receptors and/or by promoting coat assembly. Related adapters are also involved in forming nonclathrin-coated vesicles in intra-Go@ transport [32*]. It is tempting to speculate that adapter-like molecules may also drive the selective formation of basolateral (and even apical) transport vesicles. To dissect out the components of the sorting machinery, functional assaysin which defined steps of the targeting pathway are reconstituted must be developed, as has been used for the study of vesicular transport along the early biosynthetic pathway (see Waters, GriE and Rothman, this issue, pp 615-620). One promising approach is to mechanically permeabilize the apical surface, generating holes that are large enough for vesicles to escape from the cells. Sorting from the TGN has been reconstituted in such a system, leading to the purification of apitally and basolaterally targeted vesicles. They exhibit par tially overlapping compositions, but a number of proteins unique to each class of vesicle are good candidates for components of the sorting machinery [33e*]. Recycling and tmnscytosis from basolateral endosomes have also been reconstituted in similar semi-intact systems. Both processes require cytosolic proteins (M Bomsel and K Mostov, unpublished data) [34*]. Altematieiy, smaller holes can be made in either cell surface with the bacterial toxin streptolysin-0, allowing the reconstitution of multiple steps of membrane traffic. TGN-derived vesicles do not leak out from the permeabilized cells and instead fuse with the plasma membrane in a process also requiring cytosolic proteins [35*]. Deliveiy is inhibited by the non-hydrolyzable GTP analogue,

GTP-yS, suggesting that a GTPase is involved, as in many other membrane traIEc events (see Goud and McCaIfrey, this issue, pp 626-633), but no epithelial-specific examples of GTPases have yet been reported [ 36.1. A third approach uses cell-free systems. Various carrier vesicles have been purified from rat liver, either by budding from TGN isolated on a solid support or by cell fractionation and immunoadsorption [37*,38’]. Again unique components of these vesicles may be part of the sorting machinery. These reconstituted systems have the major advantage of allowing the manipulation of the components essential for a single step of transport to occur and offer access to cytosolic machinery involved in sorting.

Once formed, the different vesicles presumably require sorting information to travel to the correct cell surface. Directed transport may involve interaction with the cytoskeleton, which displays a highly polarized distribution in epithelial cells [39,40]. Pathways to the apical surface from both the TGN and basolateral endosomes are greatly facilitated by microtubules. In contrast, delivery to the basolateral surface either directly from the TGN or from the apical endosomes (by transcytosis) is much less dependent on microtubules [40,41*-44*]. In both cases, a direct involvement of mechanochemical motors remains to be demonstrated formally. However, both dynein and kinesin are required for the delivery of material from separate early apical and basolateral endosomes into a common prelysosomal compartment [7**].

Do endosomes and the trans-Golgi use similar machinery?

network

The TGN and endosomes are the two key sites for sorting decisions to take place. Do they recognize the same sorting signals and use the same machinery [3=*]? This issue is especially acute for apical sorting because this process varies with cell type. In hepatocytes, the machinery that specifies apical sorting might be located exclusively in endosomes, while in MDCK and CaCo2 cells the same apical sorting machinery might be located both in endosomes and in the TGN. The expression in different cell types of the same protein with an ultimate apical localization supports this hypothesis. Indeed, aminopeptidase N and dipeptidyl peptidase IV use the direct TGN-to-apical surface pathway in MDCK cells, are ba.solateraIly deIivered and then transcytosed in hepatocytes, and follow both pathways in CaCo2 cells (J Casanova and K Mostov, unpublished data) [ 46.1. However, there are three major diIferences in sorting between the TGN and endosomes. First, clustering of glycolipids and GPI-anchored proteins is believed to play a key role in sorting from the TGN to the apical surface [3**]. They cannot play such a role in endosomes, because at least in CaCo2 cells they are not transcytosed [ 18*,47**]. It will be interesting to determine how GPIanchored proteins and glycolipids reach the apical surface of hepatocytes. Second, GTP-yS inhibits TGN-to-

Sorting

of plasma

apical targeting in MDCK cells, and, at least in non-polarized cells, inhibits budding from the TGN [35*,48*,49], Ln contrast, GTP-yS stimulates budding of transcytotic vesicles containing p&R (M Bomsel and K Mostov, unpublished data). Finally, the mutation of the Ser phosphorylation site of the p&R to an Asp results in a molecule that is delivered directly from the TGN to the basolatetal surface. Subsequently, in the endosome, this mutant is efficiently targeted to the apical surface. The negative charge on the Asp is used as an apical targeting signal by the endosome, but is ignored by the TGN, so these compartments must rely on different signals.

membrane

examples

of polarized

sorting

Information about the sorting machinery can be obtained by studying how the epithelial phenotype is generated [ 1,50**], because in the developing epithelium, the nonpolarized precursor cell has to elaborate the sorting machinery de nova. This multistage process first requires the expression of cell adhesion molecules (CAMS). Interestingly, cell-cell and cell-substratum interactions quickly induce apical pole formation, while the generation of the basolateral pole occurs more slowly [ 1). Transcytosis, which appears soon after cell-cell contact, may actively participate in the further establishment of polarity (A Pollack and K Mostov, unpublished data). Recent evidence suggests that tight junctions are not a prerequisite for the establishment of polarity, as a CAM expressed in a nonpolarized cell is sufficient to induce the appearance of polarized cell surface domains [50**]. Similarly, polarized plasma membrane domains (whose boundaries are de&-red by unknown mechanisms) exist in many non-epithelial cells, such as neurons, sperm, and lymphocytes [51,52*]. In neurons, proteins found at the apical epithelial surface, including GPI-anchored proteins, are located only in axons and are excluded from the soma and dendrites [53**]. This suggests that the principles of sorting in epithelial cells may apply to other pdarized cell types.

Conclusion The detailed description of the polarized organization of cells in various epithelia and the multiple pathways that membrane proteins can use has been the major focus of research in this field in past years. In addition, recombinant DNA technology has yielded considerable knowledge of the signals for polarized sorting. Understanding the sorting machinery requires biochemical and genetic analysis, an endeavor that is just beginning.

in epithelial

cells Bomsei

and Mostov

Acknowledgements We thank members of the Mostov laboratory for stimulating discussion, Michael Roth and Enrique RodriguezBoulan for permission to cite unpublished work, and Maria Kerschen for preparing the manuscript. This work was supported by grants from NE-I, the Searle Scholars Program, and the Cancer Research Institute to Keith Mostov.

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WANDINGER-NESS A, BENNET MK, ANTONY C, S~MONS K: Distinct Transport Vesicles Mediate the Delivery of Plasma Membrane Roteins to the Apical and Basolateral Domains of MDCK Cells. J cell Biol 1990, 111:987-1000. The latest in a series of papers using the perforated ceU system to produce TGN-derived vesicles from MDCK cells. Vesicles targeted to the apical and basolateral surfaces were separately purified and had overlapping but distinct compositions. These unique proteins may be part of the sorting machinery. ..

34.

PODBIIEWI~Z

.

for Transfeti Cells. EMBO

Endocytosis in perforated

B, MEW

RecycIing

1: ATP and in Intact

Cytosol Requirements and Disrupted MDCK

J 1990, 93477-3487. and basolateral recycling of nansfenin MDCK cells. Recycling requires ATP

35.

GRAVOTTA

.

Iluetua

HA

were reconstituted and cytosol.

DM, ADESNIK M, SABAT~NI DD: Transport thorn the trunsGolgi Network to the

of

In-

ApicaI

Sorting

of plasma

Surface of MDCK Cells Permeabilixed ln Their Basolateral Plasma Membranes: Energy Dependence and lnvolvement of GTP-binding Proteins. / Cell Biol 1990, 111:28932908. Streptolysin-0 was used to permeabilize MDCK cells and transport of HA, the influenza hemagglurinin protein, to the apical surface was reconstituted. GTPTS blocks apical transport and must be added eariy to exert its effect. 36. .

CHA~RIER P, PARTON RG, HAURI HP, SIMONS K, ZE~IAL M: Localixation of low Molecular Weight GTP Binding Proteins to Exocytic and Endocytic Compartments. Cell 1990, 62:317-329. Seved GTPases of the Rab famiiy were cloned from MDCK ceil cDNA and these proteins localized to distinct compartments such as eariy endosomes and the prelysosomal component. No epithelial-specific GTPases were found. KE: Exocytic Transport Vesicles Generated In Vitro from the TransGolgi Network Carry Secretory and Plasma Membrane Proteins. froc Null Acad Sci USA 1990, 87:7717-7721. Golgi stacks were isolated from liver by immunoadsorption. In a celifree system, specific carrier vesicles were budded off from the TGN.

37. .

SALAMERO J, SZTLIL ES, How~u.

Tratfic Between Distinct Plasma Membrane Domains: Isolation and Characterization of Vesicular Carriers Involved in Transcytosis. Cell 1991, 64:81-89 Vesicles carrying p&R were purified from rat Liver by ceii fractionation and immunoadsorption. These vesicles were enriched in a novel 108 kD protein, which may be pan of the transcytotic sorting machinery. 38. .

39.

SZ~UL E, KApLIN A, SAXAN

L, PARADE G: Protein

BACAUAO RC. ANTONS C. Darn C, KARSENI-I E, STEIZER EHK. SIMMONS K: The SubceUubr Organization of Madin-Darby Ca-

nine Kidney Cells During the Formation ithelium. / Cell Biol 1989, 109:2817-2832. 40.

of a Polarized

Ep-

ACHLER C, FINER D, MEATE C, DRENCKHAHN D: Role of Microtubules in Polarized Delivery of Apical Membrane Proteins to the Brush Border of the Intestinal Epithelium. / Cell Biol 1989, 109z179189.

41. .

HUMMER W, M,i LE P. MEUMAN I: Ditferential Microtubule Requirements for Transcytosis in MDCK Cells. EMEO / 1990, 9:3515-3525. The microtubule requirement for transcytosis was investigated in MDCK cells. Basolaterai-to-apicai transcytosis of p&R was inhibited by nocodazole. In contrast, the apical-to-basolaterai transcytosis of IgG by the FCRB was not sensitive to nocodazole. 42. .

Basrm~to PP. MCKINNON WC, MOSTOV KE: Effect of Nocodazole on Vesicular Traffic to the Apical and Basolateral Surfaces of Polarized MDCK Cells. J C&Y Biol 1990, 111:2365-2373. The study of the involvement of the microtubuiar network was extended to the other pathways of membrane traffic. Using the wild-type and mutant p1gR.s expressed in MDCK ceiis as marker, nocodazole was found to inhibit all apically targeted pathways, but did not affect base. laterally targeted pathways.

43. .

K, BUCHER K, HAURJ H-P: Microtubule Perturbation Retards both the Direct and the Indirect Apical Pathway but does not Alfect Setting of Plasma Membrane Proteins in Intestinal Epithelial Cells (CaCo-2). EMBOJ 1990, 9:31633170. In CaCo2 cells; antimicrotubule drugs inhibited delivery to the apical surface from both the TGN and basolateral endosomes.

44. .

M+I-I-ER

GUEE~ T. LEBMc A Nbular Otganixation Patbways of Plasma nal Epithellal Cells. This paper is very similar to of the details are ditferent

QUARONI A, RODRIGUEZ-BOUIAN E: Microand its Involvement in the Biogenetic Membrane Proteins in CaCo-2 Intesti/ Cell Bid 1991, 113:27>288. that by Matter ef al 143 *I, although some

membrane

proteins

in epithelial

cells Bomsel

and Mostov

45.

Ensas U, KLIJMPERMAN J, HAm H-P: Nocodazole, a Microtubule-active Drug, Interferes witb Apical Rotein Delivery in Cultured Intestinal Epitbellal Cells (CaCo-2). / CeU Biol 1989, 108:1322.

46.

WEVZB NOREN

.

H-P, HANSEN GH, FUHRIR 0, SPEI~~ M: Aminopeptidase

C, IDOK AT, SJEISIROM H, N is Directly Sotted

to the Apical Domain in MDCK Cells. / cell Bid 1990, 111:2932-2930. Aminopeptidase N is normally basolateraUy delivered and then transcyto.sed to the apical surface in hepatocytes. When the protein is exoge nously expressed in MDCK cells, it is delivered directly from the TGN to the apical surface. Similar unpublished experiments have been carded out by Casanova and Mostov, using dipeptidyl peptidase Iv. 47. ..

VAN’T HOF W, VAN MEER G: Generation

of Lipid Polarity in Intestinal Epithelial (CaCo-2) Cells: Sphingolipid Synthesis in the Golgi Complex and Sorting Before Vesicular Tratfic to the Plasma Membrane. / cell Bid 1990, 111:977-986. ln CaCo2 cells. giycolipids are directly transported from the TGN to their correct surface. Transcytosis of lipids is very slow and quantitatively unimportant. This work completed the description this same group gave of the Lipid u-a& in other epithelia, mainly in MDCK cells.

48. .

TOOZE SA, WEISS U, HUITNER WB: Requirement for GTP Hydrolysis in the Formation of Secretory Vesicles. Nature 1990, 347:207-208. Both regulated and constitutive TGN-derived vesicles can be tonned in a celifree system. These processes are inhibited by GTPyS. 49. .

MIUER SG, MOORE H-PH: Reconstitution of COnStiNtiVe cretion Using Semi-intact Cells: Regulation by GTP but Calcium. J Cell Biol 1991, 112:3+54. Streptolysin-0 was used to pcrmeabilize non-polarized cells and livery from the TGN to the cells surface was reCOnStiNted. This inhibited by GTP-fi, but not by calcium depletion,

Senot dewas

50. ..

MCNEIU H, OZAWA M, KEMIER R, NELSON WJ: Novel Function of the Cell Adhesion Molecule Uvomotulin as an Inducer of CeU Surface Polarity. GelI 1990, 62:309-316. Transfecting the ceU adhesion molecule uvomorulin into fibroblasts converted them to an epitheliai-like morphology, even in the absence of tight junctions. Na+-K+-ATPase was localized to the sites of cell-cell contact.

51.

FRIEND DS: Morphology of Mammalian Sperm Membranes During Dlffetentiation, Maturation, and Capacitation. J Elec Micro Tech 1990, 16:281-297 A review of the morphology of sperm, which have five distinct plasma membrane domains. BEARER El

KUPFER A, MOSMANN TR, KUPFER H: Polarized Expression of Cytokines in CeU Conjugates of Helper T Cells and Splenic B Cells. Proc Natl Acud Sci USA 1991, 88:775-779. One of several demonstrations that normally non-polarized lymphocytes develop polarity when interacting with other cells. 52. .

CG, PARTON RG, SU.IONS K Polarized Setting of Glypiated Proteins in Hippocampal Neurons. Nafure 1991, 349:158-161. These authors had previously demonstrated that the influenza HA protein, which is apically targeted in epithelia, is conUned to axons of neurons. Conversely, the vesicuiar stomatitis virus G protein, which is basolaterally targeted in epithelia, is found in the soma and dendrites in neurons. Here, they show that GPI-anchored proteins, another apicaUy targeted protein in epithelia, are found only in the axons of neurons. An analogy between axons of neurons and the apical domain of epithelial ceU is proposed.

53. ..

Darn

M Bomsel, Etats Ii&s Mol&ulaires, ER61, CNRS, 45 Rue des Saints-Peres, 75006 Pads, France. K Mostov, Department of Anatomy, University of California at San Francisco, Box 0452, San Francisco, California 94143, USA.

653