Plasma membrane protein sorting in epithelial cells: Do secretory pathways hold the key?

TIBS 13-May 1988

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in The Enzymes 3rd Ed., Vol. IIA, (Boyer, P. D., ed.), pp. 369-396, Academic Press Rossman, M. G., Liljas, A., Branden, C-I. and Banaszak, L. J. (1975) in The Enzymes 3rd Ed., Vol. IIA (Boyer, P. D., ed.), pp. 61-101, Academic Press Birktoft, J. J. and Banaszak, L. J. (1984) in Peptide and Protein Reviews Vol. 4 (Hearn, M. T. W., ed.), pp. 1-46, Marcel Dekker Holbrook, J. J., Liijas, A., Steindel, S. J. and Rossmann, M. G. (1975) in The Enzymes 3rd Ed., VoL IIA, (Boyer, P.D., ed.), pp. 191292, Academic Press Birktoft, J. J. and Banaszak, L. J. (1983) J. Biol. Chem. 258,472-482 Clarke, A. R., Smith, C. J., Hart, K. W.,

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Wilks, H. M., Chia, W. N., Lee, T. V., Birktoft, J. J., Banaszak, L. J., Barstow, D . A . , Atkinson, T. and Hoibrook, J. J. (1987) Biochem. Biophys. Res. Commun. 148,15-33 Fernley, R. T., Lentz, S. R. and Bradshaw, R. A. (1981) Biosci. Rep. 1,495-507 Srere, P. A. (1972) in Energy Metabolism and the Regulation of Metabolic Processes (Mehlman, M. A. and Hanson, R. E., eds), pp. 79-91, Academic Press Beeckmans, S. and Kanarek, L. (1981) Fur. J. Biochem. 117, 527-535 Sumegi, B. and Stere, P. A. (1984) J. Biol. Chem. 259,15040-15045 Margulis, L. S. (1970) Origin of Eucaryotic

Plasma membrane protein sorting in epithelial cells: Do secretory pathways hold the key? James R. Battles and Ann L. Hubbard The Madin-Darby canine kidney (MDCK) cell sorts newly synthesized apical and basolateral plasma membrane proteins intracellularly and then ships them directly to the correct plasma membrane domains, whereas the rat hepatocyte sorts apical and basolateral proteins after their arrival at the basolateral domain of the plasma membrane and then employs a transcytotic mechanism to deliver the apical proteins to the apical domain. This difference in plasma membrane protein sorting palhways may relate to the observation that the hepatocyte, unlike the MDCK cell, does not have an apically directed secretory path way. Most epithelial cells are organized into extensive arrays which form boundaries between two different environments. The epithelial cells exhibit an intrinsic polarity, presumably to mediate interactions with and between these two environments. This asymmetry is particularly evident in the case of the epithelial cell plasma membrane, which can be thought of as being divided into specialized regions called domains: (1) the apical or luminal domain, comprising the 'free' surface; (2) the lateral domain, tightly apposed to the lateral domains of adjacent epithelial cells; and (3) the basal or serosal domain, resting on a basal lamina and in contact with the circulation usually via an intervening layer of connective tissue 1. On the basis of observations of a J. R. ,,lartles is at the Department of Cell Biology and AJ~atomy, Northwestern University Medical School, 3o3 East Chicago Avenue, Chicago, IL 60611, t'ISA. A. L. Hubbard is at the Department of Cell Biology and Anatomy, Johns Hopkins Uniwtrsity School of Medicine, 725 North Wolfe Street, Bdt~imore, MD21205, USA.

number of epithelial cell systems, it is now apparent that there is a striking molecular correlate to this structural and functional polarity of the epithelial cell plasma membrane: in many cases, the different plasma membrane domains have been shown to contain distinct complements of integral membrane proteins (receptors, transporters, enzymes, adhesion molecules, etc.) whose asymmetric distribution ensures that their specific functions are performed at the different surfaces of the epithelial cell ~. Since virtually all of the domain-specific plasma membrane proteins studied to date are integral membrane glycoproteins with complex-type asparagine-linked oligosaccharides, they presumably have in common many steps in the initial stages of their life cycles, including: (1) cotranslational insertion into the membrane of the rough endoplasmic reticulum coincident with the receipt of the high mannose precursor form of the asparagine-linked oligosaccharides; and (2) vesicular transport from endoplasmic reticulum to the cis part of the Golgi complex, through the Golgi com-

Cells, Yale University Press 19 Steinman, H. M. and Hill, R. L. (1973) Proc. Natl Acad. Sci. USA 70, 3725-3729 20 Nishiyama, M., Matsubara, N., Yamamoto, K., lijima, S., Uozumi, T. and Beppu, T. (1986) J. Biol. Chem. 261,14178-14183 21 Rosenkrantz, M., Alam, T., Kim, K., Clark, B. J., Stere, P. A. and Guarente, L. P. (1986) Mol. Cell. Biol. 6, 4509-4515 22 Obaru, K., Nomiyama, H., Shimada, K., Nagashima, F. and Morino, Y. (1986)J. Biol. Chem. 261,16976--16983 23 Wu, M, and Tzagoloff, A. (1987) J. Biol. Chem. 262,12275-12282 24 McAlister-Henn, L. and Thompson, L. M. (1987)J. Bacteriol. 169, 5157-5166

plex in a cis-to-trans direction - coincident with the processing of the asparagine-linked oliogosaccharides to their complex (terminally glycosylated) forms - and eventually to the plasma membrane 2. How do epithelial cells sort these proteins to their respective plasma membrane domains? IntraceHular sorting in the MDCK cell Until now, most of our knowledge regarding the mechanisms of plasma membrane protein sorting in epithelial cells has been acquired from studies of the Madin-Darby canine kidney (MDCK) cell line. These cells exhibit all of the properties of polarized epithelial cells in culture: they grow to form a tightly sealed monolayer, and they exhibit the expected structural and functional polarity ~'3. When infected with certain enveloped RNA viruses, viral progeny bud from MDCK cells in a domain-specific fashion, e.g. influenza virions bud exclusively from the apical surface and vesicular stomatitis virions bud exclusively from the basolateral surface t'3. This polarity of virus budding reflects the domain-specific delivery of the respective viral envelope glycoproteins, HA and G protein, to the MDCK cell plasma membrane (Table I) 1'3. As a result, these viral glycoproteins have now been employed as model apical and basolateral proteins in innumerable studies of nlasma membrane protein sorting L4. One of the primary advantages for considering the behavior of these exogenous integral membrane proteins is that the absence of any pre-existing pool makes it relatively easy to monitor the progress of the initial wave of viral proteins, whether using biochemical or morphological techniques. On the basis of the data that have emerged, it is clear that HA and G travel the biosynthetic pathway together until they reach the trans-most portion of the ~) 1988.ElsevierPublicationsCambridge 0376-5067188/$02.00

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Table L Plasma membrane proteins

Cell type

Protein

Domain localization

Refs

MDCK cell

Influenza HA ~ Vesicular stomatitis virus G protein~ Na +, K+-ATPase Aminopeptidase N Dipeptidylpeptidase IV HA4 CE9 Asialoglycoprotein receptor

Apical Basolateral Basolateral Apical Apical • Apical Basolateral Basolateral

1,3--5 1,3-5 1,3,6 1,7 1,7 7 7 1,7

Direction secreted

Refs

Hepatocyte

aExogenous

Table 11. Constitutive secretory proteins

Cell type

Protein

MDCK cell

81 kDa glycoprotein Laminin Heparan sulphate proteoglycan aeu-globulin (liver form) a

Hepatocyte

Albumin Transferrin a2,.-globulin

aExogenous

Golgi complex, the trans-Golgi network; they are then sorted from one another and shipped directly to the correct plasma membrane domains (Fig. 1) TM. Experiments in which constructs containing the cDNAs encoding HA and G have been used to transfect MDCK cells have indicated that this sorting pathway is utilized for these two proteins independent of viral infection.s Similarly, there is evidence that one endogenous basolateral protein of the MDCK cell, the Na +, K+-ATPase, might also be sorted intracellularly6, but the sorting of an endogenous apical plasma membrane protein in these cells has not yet been examined. Another route discovered in hepatocytes We recently compared the sorting pathways utilized by several endogenous integral plasma membrane proteins in a different polarized epithelial cell, the rat hepatocyte 7. The group of proteins examined included three apical proteins (aminopeptidase N, dipeptidylpeptidase IV and a ---105 kDa protein called HA 4) and two basolateral proteins (a --48 kDa protein called CE 9 and the asialoglycoprotein receptor) (Table I). These experiments were performed in vivo and employed pulsechase radiolabeling with l-[35S]methionine in conjunction with subcellular fractionation and specific protein

Apical Basolateral Basolaterai Apical and basolaterai Basolateral Basolateral Basolateral

9-11 9 9 11 12,13 12,13 12,13

experiments; and (4) they were oriented in an ectoplasmic-side-out fashion, as expected for vesicles derived from the plasma membrane but not for those derived from intraceUular organelles. At chase times greater than 45 rain, the newly synthesized apical proteins gradually began to appear in the apical plasma membrane vesicles, but they did so at different rates. The time required for half of the pulse-labeled apical proteins to move to the apical domain was estimated to be in the range of 90-120 rain for aminopeptidase N and dipeptidylpeptidase IV, but was considerably greater than 150 rain for HA 4. Our results suggest a mechanism for hepatocyte plasma membrane biogenesis in vivo in which both the apical and basolateral proteins are shipped first to basolateral domain of the plasma membrane, and then the apical proteins are sorted out and shipped to the apical domain at distinct rates (Fig. 1). Implicit in this mechanism is the notion that the hepatocyte, unlike the MDCK cell, sorts its apical and basolateral proteins following their arrival at the basolateral domain of the plasma membrane.

immunoprecipitation techniques. Briefly, we measured the rates at which the newly synthesized apical and basolateral proteins appeared in a highly What is the relationship to secretory purified preparation of intact hepato- pathways? cyte plasma membrane sheets and in Clearly, the sorting of endogenous apical and basolateral plasma mem- apical proteins in the MDCK cell must brane vesicle subfractions which were be examined before we can decide derived from the plasma membrane whether this difference in sorting pathsheets by sonication and sucrose den- ways might somehow simply reflect difsity gradient centrifugation or free-flow ferences in the handling of exogenous electrophoresis. We found that the (viral) versus endogenous plasma newly synthesized apical and baso- membrane glycoproteins. However, lateral plasma membrane proteins we presently prefer to think that the reached the hepatocyte plasma mem- observed difference is somehow brane at approximately the same rate; related to the pathways of protein the mature (terminally glycosylated) secretion operating within these two forms of each of the proteins reached cell types. Secretory proteins and its maximal specific radioactivity in the 'plasma membrane proteins are plasma membrane sheet fraction after believed to traverse the same bio45 min of chase. However, at this time, synthetic pathway, at least until they the newly synthesized proteins (apical reach the trans portion of Golgi comand basolateral alike) were present in plex 2. At this point, the secretory provesicles which appeared to be bona fide teins are either: (1) packaged without basolateral plasma membrane by a significant concentration into vesicles number of criteria: (1) they fraction- and continuously discharged by exocyated like basolateral plasma membrane tosis (the constitutive pathway) or (2) in sucrose density gradients and in free- concentrated and stored in secretory flow electrophoresis; (2) they could be granules until exocytosis is initiated by separated from the bulk of the likely some external stimulus (the regulated organellar contaminants, including pathway)2,s. vesicles derived from the trans-Golgi MDCK cells are known to constitucisternae, the trans-Golgi network and tively secrete endogenous proteins endosomes; (3) they contained the from either their apical or basolateral proven basolateral constituents CE 9 surfaces: the basal lamina constituents and the asialoglycoprotein receptor, as laminin and heparan sulphate proteojudged from vesicle immunoadsorption glycan are secreted predominantly

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Microvillus Apical ~ .... (Luminal)

Sinusoid Lateral Apical (Bile Canaliculus) Basal (Sinusoid)

" : " : ~

~:~

(Serosal)

MDCK Cell

Hepatocyte

Fig. 1. Schematic diagrams of M DCK cell and hepatocyte illustrating the differences in the pathways taken by constitutive secretory proteins (dashed arrows) integral plasma membrane proteins (solid arrows) in these two epithelial cell types. The postulated sites of sorting of apical and basolateral plasma membrane proteins are designated by asterisks. In the hepatocyte, the sorting occurs after the proteins arrive at the basolateral plasma membrane; whereas, in the MDCK cell, the proteins appear to be sorted intracellularly, probably in the trans-Golgi network, before they ever arrive at the plasma membrane. This difference may relate to the observation that the hepatocyte, unlike the MDCK cell, does not have an apically directed pathway for constitutive protein secretion.

from the basolateral surface9, whereas an 81 kDa glycoprotein is secreted predominantly from the apical surface 9-n (Table II and Fig. 1). In addition, a variety of exogenous secretory proteins introduced into MDCK cells by transfection appear to be secreted constitutively from apical and basolateral surfaces in roughly equal amounts mJ~. An interesting example of the latter is the liver form of the protein a2u-globulin, which is believed, in hepatocytes, to be secreted exclusively from the basolateral surface (Table II) 12J3. In contrast to MDCK cells, hepatocytes only have a basolateraily directed secretory pathway (Fig. 1), and this is the pathway through which the hepatocyte constitutively secretes the major plasma proteins, such as albumin and transferrin (Table If) 12'13. Those protein components of intrahepatic bile are not secreted directly into bile, but are believed to be derived from the plasma either by: (1) a paracellular route, involving passage through the intercellular space and the hepatocytes' tight junctions, or (2) by a fluid-phase or receptor-mediated transcytotic route, in which endocytic vesicles formed at the basolateral surface of the hepatocyte entrap plasma constituents, transport them through the cytoplasm and then release them into the bile canali-

cular lumen upon fusion with the apical plasma membrane ~2J3. Polymeric immunoglobulin A is a good example of a protein which is transported from plasma to bile by rat hepatocytes using such a receptor-meditated transcytotic pathway ~2J3. That hepatocytes do not express an apically directed secretory pathway could at least partially explain why they need to use a similar basolateral-to-apical transcytotic pathway for the delivery of their apical plasma membrane proteins 7. Future experimentation

Does this apparent relationship between the exocytic pathways operating within the MDCK cell and within the rat hepatocyte apply to other epithelial cell types as well? Besides the rat hepatocyte and the MDCK cell, no other epithelial cell has yet been examined in sufficient detail. While there is some contradictory evidence available on plasma membrane protein sorting in the enterocyte ~4-16, virtually nothing is known about the secretory pathway(s) of this cell. Likewise, recent experiments suggest that cells of the enterocy~e-like Caco-2 line have a basolaterally directed secretory pathway and show the usual domainspecific budding of influenza and vesicular stomatis virions 17, but the sorting

pathways for these viral proteins or any endogenous plasma membrane proteins have yet to be elucidated. Clearly, there is a need to compare directly the sorting of endogenous plasma membrane proteins and secretory proteins in diverse epithelial cell types. Is there some built-in directionality in the pathways of all exocytic vesicular traffic occurring within a single epithelial cell type, such as that which could arise from restricting the spatial distributions of microtubules (or perhaps other cytoskeletal elements as well) which have been postulated to serve as 'tracks' to support vesicular traffic within cellsSjS? In this regard, it is interesting to note that microtubuledisrupting drugs (such as colchicine) appear to impair plasma membrane protein sorting in both MDCK cells19 and rat hepatocytes (Ref. 20, and Stieger, B. and Hubbard, A. L., unpublished). Should we expect that similar mechanisms will be used to sort the plasma membrane and constitutive secretory proteins in a given epithelial cell type? Recent evidence indicates that regulated secretory proteins and plasma membrane proteins are sorted in the trans-most cisterna of the Golgi complex or the trans-Golgi network 2~. Are newly synthesized plasma membrane

184 proteins and constitutive secretory proteins sorted in a similar fashion, or do they travel to the cell surface within the same post-Golgi vesicles? Albumin and transferrin, both constitutive secretory proteins, and the vesicular stomatis virus G protein, an exogenous plasma membrane protein, have been colocalized in putative secretory vesicles in cells of the Hep G2 human hepatoma line 22, but there is no indication as to whether these proteins are headed into or out of the cell, and the Hep G2 cell is not a polarized cell. More recent morphological studies have suggested that the constitutive secretory protein albumin may actually be sorted from the asialoglycoprotein receptor, an endogenous plasma membrane protein, in the trans-Golgi network of the rat hepatocyte 23. In MDCK cells, lysosomotropic amines have been shown to exert differential effects on the sorting of constitutive secretory proteins and plasma membrane proteins: while treatment with ammonium chloride brings about a change in the direction of laminin and heparan sulphate proteoglycan secretion from basolateral to both apical and basolateral 9, it does not cause detectable mis-sorting of the exogenous apical plasma membrane protein influenza HA (Ref. 24) or the endogenous basolateral plasma membrane Na+,K+-ATPase 6. Perhaps this means that plasma membrane proteins and constitutive secretory proteins are indeed transported to the cell surface in different post-Golgi vesicles and that they are sorted by different mechanisms, ones which are differentially sensitive to ammonium chlorideinduced changes in intraluminal pH. It seems that this issue of post-Golgi localization for newly synthesized plasma membrane and constitutive secretory proteins could be easily addressed for the rat hepatocyte in vivo using pulsechase radiolabelling and subceUular fractionation techniques (such as vesicle immunoadsorption) to compare the pathway taken by newly synthesized albumin or transferrin to that of the hepatocyte plasma membrane proteins. The most plausible model for the sorting of membrane and secretory proteins in cells invokes the existence of specific receptors which can recognize certain structural features or 'signals' on the proteins to be sorted and thereby direct them to the specified cellular locations 25. That the hepatocyte and the MDCK cell appear to sort their plasma membrane proteins along

T I B S 1 3 - M a y 1988

different pathways may indicate that the relevant sorting signals and receptors will prove to be epithelial cell typespecific as well. Such an intriguing possibility mandates detailed comparisons of the sorting pathways taken by various plasma membrane proteins (and probably some additional secretory proteins l°'n as well) when they are expressed in heterologous epithelial cell types. For example, one could infect rat hepatocytes with influenza virus in vivo, or in isolated perfusion, and compare the sorting of H A and the endogenous apical proteins. Alternatively, one could examine the sorting pathway taken by an apical plasma membrane protein of the hepatocyte when introduced into MDCK cells by transfection. A good candidate for this type of study might be the apical hepatocyte plasma membrane protein H A 4 (Ref. 7) which is not detected in large amounts in rat kidney 26. Also potentially valuable would be detailed structural comparisons of analogous plasma membrane proteins expressed by these and other epithelial cell types, preferably those within a single species, with attention to amino acid sequence differences or to any co- or posttranslational modifications which might serve as cell-type-specific sorting signals. A good place to start would be to compare the endogenous apical plasma membrane protein aminopeptidase N isolated from rat river and kidney or from canine hepatocytes and MDCK cells ~. Adoption of such a multifaceted approach will undoubtedly begin to yield insight into the details of the sorting mechanisms. Acknowledgements

We gratefully acknowledge the important contributions, both conceptual and experimental, of Drs Helene M. Feracci and Bruno Stieger; and grant support from the National Institutes of Health. References

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2 Farquhar, M. G. (1985)Annu. Rev. CellBiol. 1,447-488 3 Rodriguez-Boulan,E. (1983) in Modern Cell Biology Vol. 1 (Satir, B. H., ed), pp. 119-170, Alan R. Liss 4 Matlin,K. (1986)J. CellBiol. 103,2565-2568 5 Gottlieb, T. A., Gonzalez, A., Rizzolo, L., Rindler, M. J., Adesnik, M. and Sabatini, D. D. (1986)J. CellBiol. 102,1242-1255 6 Caplan, M. J., Anderson, H. C., Palade, G. E. and Jamieson, J. D. (1986) Cell 46, 623631 7 Bartles, J. R., Feracci, H. M., Steiger,B. and Hubbard, A. L. (1987) J. Cell Biol. 105, 1241-1251 8 Burgess,T. L. and Kelly, R. B. (1987)Annu. Rev. CellBiol. 3,243-293 9 Caplan, M. J., Stow, J. L., Newman, A. P., Madri, J., Anderson, H. C., Farquhar, M. G., Palade, G. E. and Jamieson, J. D. (1987) Nature329,632-635 10 Kondor-Koch, C., Bravo, R., Fuller, S. D., Cutler, D. and Garoff, H. (1985) Cell 43, 297-306 11 Gottlieb, T. A., Beaudry, G., Rizzolo, L., Colman, A., Rindler, M., Adesnik, M. and Sabatini, D. D. (1986) Proc. Natl Acad. Sci. USA 83, 2100-2104 12 Kloppel, T. M., Brown, W. R. and Reichen, J. (1986)Hepatology6, 587-594 13 Coleman, R. (1987)Biochem. J. 244,249-261 14 Massey,D., Feracci, H., Gorvel, J-P., Rigal, A., Soulie, J. M. and Maroux, S. (1987) J. Membr. Biol. 96,19-25 15 Danielsen, E. M. and Cowell, G. M. (1985) Fur. Z Biochem. 152,493-499 16 Lorenzsonn, V., Korsmo, H. and Olsen, W. A. (1987)Gastroenterology92, 98-105 17 Rindler, M. and Traber, M. G. (1987)1. Cell Biol. 105(4,Pt. 2), 58a (Abstr.) 18 Vale, R. D., Schnapp, B, J., Mitchison, T., Steuer, E., Reese, T. S. and Sheetz, M. P. (1985) Cell43, 623-632 19 Rindler, M. J., Ivanov, I. E. and Sabatini, D. D. (1987)Z Cell Biol. 104,231-241 20 Durand-Schneider, A-M., Maurice, M., Dumont, M. and Feldmann, G. (1987)Hepatology 7,1239-1248 21 Orci, L., Ravazzola, M., Amherdt, M., Perrelet, A., Powell, S., Quinn, D. L. and Moore, H-P.H. (1987)Cell51,1039-1051. 22 Strous, G. J. A. M., Wiilemsen, R., van Kerkhof, P., Slot, J. W., Geuze, H. J. and Lodish, H. (1983)J. CellBiol. 97,1815-1822 23 Geuze, H. J., Slot, J. W. and Schwartz, A. L. (1987)J. CellBiol. 104,1715-1723. 24 Matlin, K. S. (1986)J. Biol. Chem. 261, 15172-15178 25 Blobel, G. (1980) Proc. Natl Acad. Sci. USA 77,1496-1500 26 Hubbard, A. L., Bartles, J. R. and Braiterman, L. T. (1985) J. CellBiol. 100,1115-1125

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