Cell, Vol. 64, 81-89,

January

11, 1991, Copyright

0 1991 by Cell Press

Protein Traffic between Distinct Plasma Membrane Domains: Isolation and Characterization of Vesicular Carriers Involved in Transcytosis Elizabeth Sztul: Adam Kaplin,t Lucian Saucan,* and George Palade* * Department of Molecular Biology Princeton University Princeton, New Jersey 08544 t Johns Hopkins University Baltimore, Maryland 21205 $ University of California San Diego School of Medicine La Jolla, California 92093

Summary We have isolated a population of vesicular carriers involved in the transport (transcytosis) of proteins from the bssolateral to the apical plasma membrane of hepatocytes. The obtalned fraction was enriched in compartments containing known transcytosed proteins and depleted in elements of the secretory pathway, Golgi elements, baeolateral plasma membrane, as well as early endosomal components. The fraction was analyzed by biochemical and Immunological procedures. Antibodies raised against the proteins in the fraction recognized a single 108K antigen. Based on its subcellular distribution, the 108K antigen may represent a novel marker for transcytotlc vesicular carriers. Introduction Polarized epithelial cells possess two morphologically and biochemically distinct plasma membrane (PM) domains that interact within distinct biochemical milieus, are composed of different lipids and proteins, and are specialized to carry out different and specific functions. In the hepatocyte, the apical PM domain faces the lumen of bile capillaries, functions in the secretion of bile compounds (Boyer, 1988), and is the terminus for the transcytotic vesicular carriers that bring polymeric IgA (pIgA) and polymeric IgA receptors (pIgA-Rs) from the sinusoidal side of the cell to the bile (Schiff et al., 1984). The basolateral domain functions in the transporter-mediated uptake of small molecules and in the receptor-mediated uptake of macromolecules such as pIgA, galactoproteins, transferrin, EGF, insulin, and lipoproteins (Wall and Hubbard, 1985; Dunn et al., 1988; Hoppe et al., 1985; Chao et al., 1981). The hepatocyte also uses the basolateral PM domain for the exocytosis of plasma proteins and lipoproteins. The existence of differentiated membrane domains implies the existence of efficient protein sorting processes. In the polarized epithelial cells so far studied, i.e., in MDCK cells (Fiindler et al., 1984; Griffiths and Simons, 1988; reviewed in Simons and Fuller, 1985; Lisanti et al., 1989) and in intestinal epithelial cells (Danielson and Cowell, 1985; Le Bivic et al., 1989), membrane proteins

appear to be directly and essentially exclusively delivered to the domain of their final functional residence from a sorting site located between the Golgi complex and the plasmalemma. (An alternative route to the cell surface has been postulated in intestinal cells [Quaroni et al., 1979a, 1979b]. Hauri and co-workers [1985] and Massey and coworkers [1987] provided evidence that apical PM proteins are transiently present in basolateral membranes.) In the hepatocyte, however, it has been proposed that biliary membrane proteins are initially inserted into the sinusoidal domain for subsequent sorting and transport to the biliary PM (Bartles et al., 1987; Bartles and Hubbard, 1988). In this case, therefore, sorting of biliary membrane proteins would occur during transcytosis. The mechanisms involved in protein removal and segregation into specific carrier vesicles have not been elucidated but, based on analogy with the identified and characterized mannose+phosphate receptor sorting system (Kornfeld et al., 1988; von Figura and Hasilik, 1986), can be assumed to depend on mutual recognition between a signal built into the primary (or higher order) structure of the protein to be targeted and a signal recognition element. Based on this paradigm, it can be proposed that distinct sorters will be involved in the selective removal of proteins destined to a common class of vesicular carriers. The mechanisms at work in piloting vesicular carriers to specific terminals remain unknown. It can be assumed, however, that distinct sorters as well as pilot molecules are specifically associated with distinct classes of vesicular carriers. Consequently, the isolation and characterization of a class of vesicular carriers can be considered as the first necessary step toward the identification of the sorter or pilot proteins performing the selection and recognition functions. In this paper we report the isolation and partial characterization of vesicular carriers involved in protein transport from the basolateral to the apical PM domain of the hepatocyte. We have used a two-step procedure to purify transcytotic vesicles: in the first step, we applied sucrose density gradient centrifugation to generate a fraction containing many classes of vesicular carriers; in the second step, we used antibodies against the endodomain of a well-characterized transcytosed protein (the pIgA-R) to specifically immunoisolate transcytotic vesicles. The isolated vesicular carriers were characterized by biochemical and immunochemical criteria. Results Fraction Enriched in mnscytotic Vesicles Can Be Prepared by Sucrose Density Centrifugatlon In our previous work, we have identified three intracellular forms of pIgA-R and have defined the time of their transit through the main compartments of the ER-basolateral PM pathway (Sztul et al., 1985a, 1985b). Using in vivo pulse-chase biosynthetic labeling, we have shown that a M, 105,000 pIgA-R form (105K) is found in rough mi-

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crosomal fractions (hence, in rough endoplasmic reticulum) 5 min after an intravenous injection of [s%+cysteine. The second form of p1gA-R (116K) appears maximally labeled at ~20 min of chase in a Golgi fraction, and the third pIgA-R form (120K) is detected in a sinusoidal PM fraction at 30 min of chase. The extracellular, secreted form of the receptor (6OK) begins to be detected in bile at ~45 min of chase. Based on these findings, we postulated that at chase times longer than 40 min, a proportion of radiolabeled cellular pIgA-R is present in transcytotic vesicles en route to the biliary PM. Consequently, transcytotic vesicles can be identified by the presence therein of radiolabeled 120K pIgA-R at the appropriate time (i.e., m45 min) after injection of [35S]cysteine. In previous cell fractionation experiments (Sztul et al., 1965b), we have used discontinuous sucrose gradients to isolate a light Golgi fraction, a Golgi heavy fraction, a crude vesicular fraction, and a residual microsomal fraction. We used the same procedure to prepare subcellular fractions from a liver of an animal sacrificed 45 min after injection of Trans[35S]label and analyzed each fraction for the content of radiolabeled pIgA-R. As shown in Figure lA, all three pIgA-R forms (identified and characterized previously) were present in the total microsomal fraction (lane TM). On the basis of our previous work, we assume that the 105K and 116K pIgA-R forms are contributed by the compartments of the ER to PM pathway, whereas the 120K pIgA-R form is present either in transcytotic vesicles or in vesicles derived from the sinusoidal PM. The light Golgi fraction contained predominantly the 116K form of the receptor (plainly visible on overexposed fluorographs), whereas the Golgi heavy fraction, besides containing 116K pIgA-R, was enriched in the 120K form. The crude vesicular fraction contained all three forms of the receptor but was most enriched for the 120K form. In the residual microsomal fraction (known to be composed of rough endoplasmic reticulum elements) we found predominantly the early, 105K precursor of pIgA-R. The preferential recovery of the 120K form of pIgA-R in the crude vesicular fraction suggests that transcytotic vesicles may account for a significant fraction of its total vesicular population. To validate this assumption, we examined whether the ligand of the pIgA-R, the dimeric IgA (dIgA), was also recovered in the same fraction. Dimeric IgA can be considered as an exclusive and most reliable marker for transcytotic vesicles. We first defined the time course of dlgA secretion into bile following an intravenous injection of biosynthetically radiolabeled dIgA. As shown in Figure 18, dlgA was seen in bile 20 min after injection and continued to be detected therein in increasing amounts for at least 20 min more, suggesting that dlgA is present within transcytotic vesicles during the length of the experiment. We therefore injected labeled plgA into a rat, sacrificed the animal 40 min later, prepared subcellular fractions (as above), and analyzed them for dlgA content. As shown in Figure lC, dlgA was enriched in the crude vesicular fraction; less was recovered in the Golgi heavy fraction and still less in the light Golgi fraction and the residual microsomal fractions. Since the highest concentrations of the ligand (dIgA) and

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Figure 1. Crude Vesicular Fraction Contains Transcytotic Vesicles

(A) A rat was injected with 1 mCi of Trans[%]label, was sacrificed 45 min later, and its liver subjected to subcellular fractionation. Approximately 300 ug of each fraction was immunoprecipitated with antipIgA-R antibodies, and the immunoprecipitates were analyzed by SDS-PAGE followed by fluorography. The 120K form of pIgA-R is enriched in the CVF. (B and C) A rat was injected with cell culture supernatant containing biosynthetically radiolabeled dlgA and was sacrificed 40 min later. (6) Bile was collected in 10 min intervals between the time of injection and the end of the experiment. Each 10 min bile sample was incubated with phosphorylcholine-Sepharose and the bound dlgA analyzed by SDS-PAGE and fluorography. HC and LC mark the positions of the heavy and light chain of dIgA. (C) The liver was removed, homogenized, and subcellular fractions were prepared as in (A). An aliquot of each fraction containing 300 ug of protein was incubated with phosphorylcholine-Sepharose, and the bound dlgA was analyzed by SDS-PAGE and fluorography. Dimeric IgA was preferentially enriched in the CVF.

of the 120K form of the pIgA-R colocalize in the crude vesicular fraction, we assume that this fraction contains transcytotic vesicles. Morphological examination of the crude vesicular fraction (data not shown), indicates a heterogeneous population of small (50-60 nm) smooth vesicles, most of them free of detectable content. Based on these biochemical and morphological results, we assume that the crude vesicular fraction contains transcytotic vesicles in addition to other classes of vesicular carriers and membrane elements; consequently, it can be used as a starting preparation for the isolation of a homogeneous fraction of transcytotic vesicles.

Transcytotic 63

Protein Traffic

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Figure 2. lmmunoisolated

Fraction Contains Transcytosed

Proteins

(A) A CVF was isolated from rat liver and subjected to immunoisolation. Aliquots of the CVF (lane SM), the nonbound material (lane NE), and the bound material (lane B) were analyzed by SDS-PAGE. Following electrophoresis. the proteins were transferred to nitrocellulose and immunoblotted with antibodies against pigA-Ft. Numbers below each lane indicate the total amount of protein loaded in that lane. (B) A rat was injected with cell culture supernatant containing biosynthetically radiolabeled dlgA and was sacrificed 40 min after the injection. A CVF was isolated from liver homogenate and subjected to immunoisolation. The CVF (55 ug, lane SM), the nonbound material (46 ug, lane NB), and the bound material (0.5 ug, lane B) were incubated with phosphorylcholine-Sepharose, and the bound dlgA was analyzed by SDS-PAGE and fluorography. The fluorograph was quantitated by densitometry. Actual readings (in arbitrary units) for SM, NB. and B fractions were 23, 13, and 63, respectively.

immunoisoiation of lkanscytotic Vesicles We (Merisko et al., 1982; Sztul et al., 1985b), and others (e.g., Devaney and Howell, 1985; Gruenberg and Howell, 1985) have shown that immunoisoiation can be used as an effective method of isolating subcellular components. Consequently, we used antibodies against the cytoplasmic domain of pigA-R (Kuhn and Kraehenbuhl, 1983; Solari et al., 1985) to isolate transcytotic vesicles, based on the assumption that the receptor must be more highly concentrated in these vesicles than in other intracellular compartments, since many other membrane proteins are left behind at different stations (especially in the sinusoidal PM) along the ER-sinusoidal PM-biliary PM pathway. The Immunolsolatad Fraction Is Enriched in 7kanscytosed Proteins The efficacy of the immunoisoiation procedure was tested by determining the distribution of known transcytosed proteins, i.e., pigA-R and digA, in derived relevant fractions. immunoisoiation was performed by incubating the immunoadsorbent (i.e., protein A-Sepharose beads coated wtth mouse IgGs against the cytopiasmic domain of pigAFt) with the crude vesicular fraction. Aiiquots of this fraction

(i.e., the starting material for the immunoisoiation), the material not bound to the beads, and the material immunoisolated on the beads were analyzed by SDS-PAGE, followed by transfer and immunoblotting with anti-pigA-R antibodies. As shown in Figure 2A, lane SM, all three forms of pigA-R were detected in the starting material (i.e., the crude vesicular fraction), with the 120K form in largest quantity. A similar but less intense pattern was visible in the nonbound fraction, as would be expected, since the immunoisoiatlon was carried out at great excess of starting material. in contrast, the bound fraction was enriched in both the 118K and the 120K pigA-R forms but was depleted in the 105K form. This result suggests that the immunoisoiation procedure is highly selective, since only a subset of pigA-R-containing vesicles was bound to the immunoadsorbent. We presume that vesicles containing the 105K form of pigA-R (“bulk flow” vesicles carrying proteins from the ER to the Golgi complex) are not immunoisoiated because the density of pigA-R in their membranes is too low or because the cytoptasmic tails of the pigA-R are masked by vesicle-associated proteins. The presence of the 118K form of the receptor in the bound fraction required further analysis. Experiments were carried out to determine whether the 118K represented receptor present in the compartments of the transcytotic pathway or in contaminating Goigi elements or in Goigi to sinusoidal PM vesicular carriers. These experiments (data to be presented) showed that the immunoisolated fraction is depleted in components of the exocytic pathway and in Golgi elements and suggested that the 116K form is present in transcytotic vesicles rather than in other contaminating compartments. This conclusion is consistent with our earlier datashowing that both the 116K and the 120K forms of pigA-R are present on sinusoidal PM at steady state (Sztui et al., 1985b). It must be pointed out that neither phosphorylation of the receptor, which is responsible for the shift from 116K to 120K (Larkin et al., 1986), nor transcytosis is iigand dependent, and so, theoretically at least, some 116K could be internalized together with the 120K pigA-R. Dimeric IgA represents the best marker for the transcytotic pathway: following internalization and an appropriate chase period, it should be preferentially recovered in transcytotic vesicles. We prepared a crude vesicular fraction from the iiverof an animal sacrificed 40 min after an intravenous injection of radiolabeled digA, subjected it to immunoisoiation, and examined the relative amounts of digA in the starting material and in the ensuing nonbound and bound fractions. As shown in Figure 28, digA was significantly (over 400-fold) enriched in the bound material, indicating that components of the transcytotic pathway were bound to the immunoadsorbent. Based on the two positive criteria we have used, i.e., the presence of the 120K pigA-R and of digA, we assume that transcytotic vesicles were preferentially isolated by the immunoadsorption procedure. To provide evidence about the degree of homogeneity of the preparation, we complemented these positive criteria by negative counter-

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Figure 4. lmmunoisolated Antigens 43 -

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Fraction Is De-enriched

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Pro-

A CVF was prepared from liver homogenate of a rat sacrificed 45 min after an injection of Transp%]label and subjected to immunoisolation. Aliquots of CVF (55 ftg, lane SM), the nonbound material (46 ttg, lane NB), and the bound material (0.5 ng, lane B) were analyzed by SDS-PAGE followed by fluorography. The bands in SM represent rapidly labeled proteins synthesized for secretion or insertion into membranes Albumin can be recognized in the first group and pIgA-R in the second. The same bands appear in the nonbound fraction. The bound fraction (B) is significantly enriched in the 120K form of p1gA-R and depleted in secretory proteins.

parts, such as the absence of secretory proteins and of proteins belonging to other subcellular compartments. The lmmunoisolated Fraction Is Depleted in Secretory Proteins To assay whether elements of the ER-sinusoidal PM secretory pathway bind to the immunoadsorbent, we performed an immunoisolation from a crude vesicular fraction prepared from the liver of an animal sacrificed 45 min after a Trans[%]label injection. This protocol results in the labeling of hepatic secretory proteins (Figure 3, lane SM) and of all pIgA-R forms (shown previously in Figure 1, lane TM). Therefore, the relative content of pIgA-R versus that of any secretory protein can be used as a measure of the specificity and efficiency of the immunoisolation. An immunoisolated fraction enriched in the 120K form of pIgA-R (presumably present in compartments of the transcytotic pathway) and de-enriched in secretory proteins (present in components of the exocytic pathway) would suggest successful immunoisolation. As shown in Figure 3, lane SM, the crude vesicular fraction contained a number of radiolabeled bands, most of which represent previously identified major secretory proteins (Sztul et al., 1983) albumin being the most dominant species. The same band pattern, in terms of number and relative intensities of bands, was detected in the nonbound fraction. A different pattern was present in the immunoisolated fraction. A dominant band, migrating with the mobility of the 120K pIgA-R (the band comigrates with

Fraction

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Is Depleted in Golgi Membrane

A CVF was isolated from rat liver homogenate and subjected to immunoisolation. Aliquots of Golgi heavy fraction (lane GH), CVF (lane SM), the nonbound material (lane NE), and the bound material (lane B) were analyzed by SDS-PAGE. Following electrophoresis, the gel was transferred to nitrocellulose and immunoblotted with antibodies raised against isolated Golgi membranes, Numbers below each lane indicate the total amount of protein loaded in that lane. Arrowheads mark proteins enriched in the immunoisolated fraction.

immunoprecipitated pIgA-R), and small amounts of albumin and other secretory proteins were detected, but the relative ratio of pIgA-R to albumin in the bound fraction was significantly higher than in the nonbound and the crude vesicular fraction. This result suggests that the immunoisolated fraction is enriched in components of the transcytotic pathway and is marginally contaminated by vesicular elements derived from the various compartments of the ER-PM pathway. The specificity of the isolation was further tested and confirmed by two criteria: pIgA-R was not recovered in the bound fraction when a nonrelated monoclonal antibody (anti-cytoplasmic domain of VSV-G) was coupled to protein A-Sepharose; and pIgA-R was not present in the bound fraction when the immunoisolation was carried out in the presence of excess soluble anti-cytoplasmic domain of pIgA-R antibody (data not shown). The lmmunoisolated Fraction Is Depleted in Golgi Membrane Antigens As shown above, secretory proteins were de-enriched in the immunoisolated fraction, suggesting that the latter does not include vesicles derived from exocytic compartments, the Golgi complex included. Since secretory proteins are more extensively labeled than membrane proteins during the pulse-chase time period tested (45 min), we tested whether the immunoisolated vesicles contained Golgi membrane proteins. In previous work, K. E. Howell and E. S. (unpublished data) raised antibodies against the membrane proteins of a rat liver fraction enriched in trans-Golgi elements and shown by immunocytochemistry that the antibodies react with antigens present in stacked Golgi cisternae. In the present study, we have used these antibodies to assay the recovery of Golgi membranes in the immunoisolated material. As shown in Figure 4, lane GH, the immune serum detected at least nine antigens in a fraction containing Golgi elements. A similar but less intense pattern was ob-

Transcytotic 85

Protein Traffic

30 Figure 5. lmmunoisolated Markers

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Is Depleted

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A CVF was isolated from rat liver homogenate and subjected to immunoisolation. Aliquots of the starting material (lane SM), the nonbound material (lane NB), and the bound fraction (lane B) were processed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with (A) antibodies against the CE-9 antigen and (B) antibodies against the a subunit of (Na+,K+)ATPase. Numbers below each lane indicate the total amount of protein loaded in that lane. Isolated sinusoidal PM fraction (Xl pg, lane PM) was analyzed as a positive control.

served in the starting material and the nonbound fractions but, of the nine proteins recognized by the antibody, only three were present in the bound fraction. Two of them migrated with mobilities identical to those of pIgA-R forms 116K and 120K (we have previously shown that the antiGolgi antibodies recognize pIgA-R [K. E. Howell and E. S., unpublished data]), and one migrated in a higher M, (220,000) range. We assume that the 220K protein was present in the antigen preparation, i.e., Golgi membranes, en route to either transcytotic vesicles or the biliary PM. Currently the identity of this antigen is not known. To further substantiate our data and to provide a more commonly used marker for the Golgi complex, we assayed galactosyltransferase activity (a marker enzyme for trans-Golgi elements [Roth and Berger, 19821) in fractions analogous to those described above. The immunoisolated fraction was approximately 187~folddepleted in galactosyltransferase activity relative to the Golgi heavy fraction and approximately 1 l-fold depleted in galactosyltransferase activity relative to the starting material and nonbound fractions. Both sets of data suggest that the immunoisolated fraction does not contain significant amounts of Golgi membranes but is significantly enriched in other membranes containing pIgA-R. The Immunokokted Fraction Is Depleted in Basokteral PM Markers The results presented above rule out the extensive presence of secretory protein carriers or Golgi-derived vesicles in the immunoisolated fraction but do not exclude its contamination by vesicles derived from other sources, especially the sinusoidal PM, with which the fraction shares common components such as the 120K and the 116K forms of pIgA-R. To test for specific sinusoidal PM antigens, we chose to follow the distribution of the CE9 antigen and the a subunit of the (Na+,K+)ATPase, two proteins previously shown to be restricted to the basolateral domain of the hepatic PM (Hubbard et al., 1985; Sztul et al., 1957). As shown in Figure 5A, the CE9 antigen was detected in the starting material and the nonbound fraction

but was absent in the immunoisolated, bound material, and as shown in Figure 58, the 96K subunit of the Na+ pump was similarly detected in the starting material and in the nonbound fraction but was absent from the bound fraction. These findings indicate that the membrane elements bound to the immunoadsorbent and containing pIgA-R are not derived from the sinusoidal PM. If vesicles derived from the sinusoidal PM (containing both pIgA-R and (Na+,K+)ATPase) were isolated, then, based on the fact that pIgA-R and (Na+,K+)ATPase are detected with roughly similar intensities in 55 ug of the crude vesicular fraction (Figure 2A and Figure 58, respectively), we would expect the signals for (Na+,K+)ATPase and pIgA-R to be of equal strength in the bound fraction. Since that is not the case, we conclude that the immunoisolation protocol selectively recovers pIgA-R carrying elements other than vesicles derived from the sinusoidal PM. Our earlier work (Meier et al., 1984) showing that pIgA-R is not a resident protein of the biliary PM (presumably because cleavage and release of the ectodomain occur immediately following insertion into that PM domain) rules out the possibility that vesicles derived from the biliary PM were present in the immunoisolated fraction and accounted for the recovery of the pIgA-R in the immunoisolated fraction. The Immunoisolated Fraction Is Depleted In Elements of the “Early” Endocytlc Pathway Since pIgA-R, with or without its ligand, follows an endocytic pathway for a part of its transcellular journey (Geuze et al., 1954; Hoppe et al., 1985) we searched for the presence of a well-established marker for early endosomes, namely the transferrin receptor (T-R) (Stoorvogel et al., 1987) in the immunoisolated fraction. As shown in Figure 6, T-R was not detected in the bound fraction in which pIgA-R was enriched at least lOO-fold, suggesting that “early” endosomal compartments are not immunoadsorbed, presumably because the antigen is less concentrated on their surfaces than on the surfaces of transcytotic vesicular carriers.

Proteins of the lmmunoisolated Fraction To examine whether a specific subset of proteins was separated by the immunoisolation procedure, equal amounts of the crude vesicular fraction were incubated with increasing amounts of immunoadsorbent. The protein patterns of the nonbound and the bound fractions from each immunoisolation were analyzed by SDS-PAGE followed by silver staining. As shown in Figure 7, the protein pattern of all bound fractions was distinct from that of the nonbound fractions. Proteins spanning the analyzed MW range were enriched in the immunoisolated fraction. The major proteins (approximate M, of 211,000, 162,000, 152,000, 140,000, and lll,OOO), preferentially concentrated in the transcytotic vesicle fraction, are marked with arrowheads. Significantly, all bound fractions had the same pattern, indicating that immunoisolation is a reproducible method of preparing the transcytotic vesicle fraction. A control lane (lane 7) in which beads without prior incubation with the crude vesicular fraction were ana-

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Figure 6. lmmunoisolated Fraction Is Depleted in Transferrin Receptor A CVF was isolated from rat liver homogenate and subjected to immunoisolation. Aliquots of CVF (lane SM), the nonbound material (lane NB), and the bound fraction (lane B) were analyzed by SDS-PAGE. Following electrophoresis, the gel was transferred to nitrocellulose and immunoblotted with a mixture of antibodies against pIgA-R and the transferrin receptor. Numbers below each lane indicate the total amount (frg) of protein loaded in that lane.

lyzed, shows only the 55K heavy and the 27K light chains of IgG. Antibodies against the lmmunoisolated Fraction The immunoisolated fraction was removed from the immunoadsorbent by solubilization in Triton X-100 and used as an antigen for polyclonal antibody production. We expected antibodies against a number of proteins, but the immune serum obtained recognized only one predominant antigen. As shown in Figure EA, a major 108K band was detected in an immunoblot of a crude vesicular fraction and a total microsomal fraction. A minor, higher M, band (M, 110,000) was also visible. The preimmune serum gave negative results for both antigens (Figure 8B).

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Figure 6. Anti-Ranscytotic Vesicle Antibodies Recognize a 1OEKAntigen Aliquots of a hepatic total microsomal fraction (200 ug of protein, lanes TM) and of the CVF (100 ag of protein, lanes CVF) were processed by SDS-PAGE and the gel transferred to nitrocellulose. The filters were immunoblotted with (A) immune serum or(B) preimmune serum. The position of the 106K antigen is marked.

To test whether the 108K antigen is restricted in its distribution (as would be expected of a transcytotic vesicle antigen) or is a component of a number of subcellular compartments, we analyzed its distribution in isolated subcellular fractions by immunoblotting. As shown in Figure 9A, the 108K antigen was not detected in a fraction containing bile canalicular PM (lane bcPM), in a fraction containing sinusoidal PM (lane blPM), nor in a Golgi light fraction (lane GL). A small amount of the 108K antigen

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Figure 7. Protein Pattern of the lmmunoisolated Fraction Increasing amounts of the immunoadsorbant (100 91, lanes 1 and 2; 200 91,lanes 3 and 4; 500 fd, lanes 5 and 6) were incubated with a constant amount of CVF lmmunoadsorbant (100 nl), no CVF added, lanes 7 and 6. Aliquots of the nonbound material (lanes NE) and of the bound fractions (lanes B) were analyzed by SDS-PAGE and the gel subjected to silver staining. Positions of proteins enriched in the immunoisolated fraction are marked by arrowheads.

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Figure 9. Subcellular Distribution of the 106K Antigen (A) Subcellular fractions were prepared from rat liver as in Experimental Procedures. Samples containing 200 pg of protein of a fraction containing bile canalicular PM (lane bcPM), basolateral PM (lane blPM), total microsomes (lane TM), and residual microsomal fraction (lane Rf) were processed by SDS-PAGE. Samples containing 100 ug of protein of CVF (lane CVF), Golgi heavy fraction (lane GH), and Golgi light fraction (lane GL) were processed by SDS-PAGE. The gel was transferred to nitrocellulose and immunoblotted with anti-IOBK antibodies. (B) ACVF was subjected to immunoisolation. Aliquots of the nonbound material (lane NB) and the bound fraction (lane B) were processed by SDS-PAGE and the gel transferred to nitrocellulose. The filter was immunoblotted with anti-106K antibodies. Four micrograms of total protein was loaded in lane B and 50 ng of total protein was loaded in lane NB.

Transcflotic 97

Protein Traffic

was visible in a Golgi heavy fraction (lane GH), and the antigen was clearly detected in a total microsomal fraction (lane TM) known to contain transcytotic vesicles, in a residual microsomal fraction (lane Rf) expected to contain newly synthesized transcytotic vesicle fraction antigens, and in a fraction enriched in transcytotic vesicles (lane CVF). The 108K antigen was not detected in serum or bile (data not shown). To test whether the 108K antigen is specific to transcytotic carriers or resides in other vesicles present in the crude vesicular fraction, we analyzed the content of the 108K antigen in relevant fractions after immunoisolation. We presumed that if the 108K antigen were a marker protein for transcytotic vesicles, it should be concentrated in the bound material as compared with the nonbound fraction. As shown in Figure 96, the antigen was preferentially enriched in the immunoisolated fraction. Comparison of Figure 96 with Figure 3 indicates that the 108K antigen is not radiolabeled following an in vivo pulse labeling and a 45 min chase. These results strongly suggest that the 108K antigen is a protein specifically associated with transcytotic vesicles. Hence, a new marker for these vesicular carriers can be added to pIgA-R and dIgA. We do not know whether the 108K antigen corresponds to the predominant silverstained band recovered in the immunoisolated fraction. The chemical characterization of this protein is the next step in this inquiry and the first necessary step toward exploring its function.

An understanding of the mechanisms that control vesicular traffic along the exocytic, endocytic, and transcytotic pathways requires the isolation of distinct classes of vesicular carriers and the characterization of their membrane proteins. We have previously examined and characterized the kinetics of transit and the compartments traversed by pIgA-R (a protein transcytosed from the basolateral to the apical PM domain) in rat hepatocytes (Sztul et al., 1985a). During that work we also identified a vesicular fraction that, based on biosynthetic labeling studies, contains carriers involved in transit between the sinusoidal and the biliary PM. Based on these studies, we devised a two-step procedure for their isolation: the first step, flotation in a discontinuous sucrose density gradient, was adapted from procedures previously developed in our laboratory (Howell and Palade, 1982) and yielded a crude vesicular fraction in which all classes of carriers appear to be present; the second step, immunoadsorption on an immobilized antibody against the cytoplasmic domain of the pIgA-R, separated transcytotic vesicles from this crude fraction. The rationale for the latter step was that only compartments containing accessible pIgA-R epitopes will interact with the immunosupport, and furthermore, that compartments containing the highest surface density of such epitopes will be preferentially bound. The isolated fraction was defined as the transcytotic vesicle fraction by relying on the presence of the mature

120K form of pIgA-R, the kinetics of its biosynthetic labeling, and the presence of its ligand dlgA as positive identifying criteria for this class of carriers. A search that us&d complementary negative criteria showed that the isolated preparation does not contain (or is significantly de-enriched in) other intracellular components such as secretory proteins, marker antigens for the sinusoidal PM, Golgi membranes, and early endosomal elements. The morphological analysis of the immunoisolated fraction showed that it consists of small, smooth surfaced vesicles, not contaminated by other recognizable hepatocytic subcellular components. We show that the transcytotic vesicle fraction contains a distinct set of proteins, not shared with other classes of vesicular carriers. We assume that the proteins present in the immunoisolated vesicles represent mostly resident, rather than cargo proteins, primarily because known cargo, i.e., the pIgA-R, is not detected by silver staining although its presence therein is well established by more sensitive procedures. A comparison of transcytotic vesicle fraction protein pattern with those already published for other classes of vesicular carriers such as clathrin-coated vesicles (Pearse and Crowther, 1987), non-clathrin coatedvesicles (Malhotra et al., 1989), vesicles derived from the trans-Golgi network (decurtis and Simon.% 1989), and membranes of mature secretory granules (Cameron et al., 1988) shows few if any similarities. The protein spectrum of coated vesicles, for instance, is dominated by clathrins (180K and 3OK) and adaptins (lOOK, 55K, and 18K) (Mahaffey et al., 1989), which are not found in the transcytotic vesicle fraction. A similar comparison with early endosomes (Schmid et al., 1988), an organelle known to be involved in early events of transcytosis (Geuze et al., 1984), reveals a number of potentially common components, but adequate identification is still needed. This type of characterization and careful comparisons with the transcytotic vesicle fraction from other sources, such as MDCK cells (Mostov and Deitcher, 1988) and neonate intestinal epithelium (Abrahamson and Rodewald, 1981; Rodewald and Kraehenbuhl, 1984), might reveal common components, presumably parts of the essential equipment required for transcytosis. Work along such lines should eventually lead to the identification of the sorters and pilots that control the activity of this class of vesicular carriers. Experimental Procedures Materials Specific biochemical compounds were purchased from Sigma Chemical Company (St. Louis, MO); Trans(35Sjlabel was from ICN Biochemicals (Irvine, CA); [3H]lJDP-galacose was from Amersham Corporation (Arlington Heights, IL). An anti-rabbit IgG-alkaline phosphatase conjugate was purchased from Promega (Madison, WI) and the alkaline phoephataae substrate system from Kierkegaard and Parry Laboratories (Gaithenburg, MD). Biosynthetically ssS-labeled dlgA (capable of binding phosphorylcholine) was a kind gift of Dr. D. Bole (Mle University School of Medicine). Antibodies against the CE9 antigen were generously provided by Dr. A. Hubbard (Johns Hopkins University School of Medicine). Antibodies against (Na+,K+)ATPase were a gift of Dr. R. Levenson (Yale University School of Medicine), and those against T-R were kindly provided by Dr. J. Woods (Yale University School of Medicine). The hybridoma cells secreting anti-plgAR cyto-

Cdl 88

plasmic domain IgGs (SC 166) (K6hn and Kraehenbuhl, 1963) were kindly provided by Dr. J.-P Kraehenbuhl (ISREC, Lausanne, Switzerland). Supernatants of cells cultured for 3 days were concentrated 25-30 times using an Amicon Centriprep device (Amicon, Danvem, MA).

In Vlvo Labeling Experiments Male Sprague-Dawley rats (120-160 g) were anesthetized with intraperitoneally injected Nembutal (Abbott Laboratories, North Chicago, IL) at 0.1 ml per 100 g of animal weight. They were then injected into the saphenous vein with either Trans[35S]label or a solution of biosynthetically labeled dIgA. At various times after injection, the livers were removed for further processing. In some experiments, bile was collected (under anesthesia) via a canula (PE 50 tubing; Clay Adams, Parsippany, NJ) inserted into the common bile duct,

Subcellular Fractlonatlon and Enzymatic Assay Homogenization of the liver and preparation of a total microsomal pellet was as described in Sztul et al. (1963). Golgi fractions and a fraction enriched in transcytotic vesicles were isolated by a modification (Howell and Palade, 1962) of the procedure of Ehrenreich et al. (1973) omitting in all cases ethanol administration to the animals. Briefly, total microsomal fractions (~120 mg of protein) resuspended to 10 ml in 1.22 M sucrose were loaded at the bottom of a discontinuous sucrose gradient (1.15 M, 0.86 M, 0.25 M), which was centrifuged for 3 hr at 82,500 x gaV in an SW27 rotor. The band that formed at the 0.251 0.66 M interface was designated the Golgi light fraction; that found at the 0.86/1.15 M interface constituted the heavy Golgi fraction; and the fraction recovered within the 1.15 M region of the sucrose density gradient was designated crude vesicular fraction. The residual microsomal fraction remained at the bottom of the centrifuge tube after the flotation of other components. These fractions have been characterized previously (Sztul et al., 1985b). Canalicular and basolateral liver plasma membrane fractions were isolated as described previously (Meier et al., 1984). Galactosyltransferase activity was measured by a modification of the method of Saraste et al. (1986) with ovalbumin as acceptor. The assay mixture (100 ul) contained 50 ul of sample, ovalbumin at 7 mglml, 2 mM ATR 200 mM MgCIz, 0.2% Triton X-100, and 50 mM Tris-HCI (pH 6.8). The assay was started by the addition of 0.76 nmol of [3H]UDP-galactose in 20 ul of HzO. The reaction was stopped by adding ice-cold 24% trichloroacetic acid. The precipitate was pelleted and washed three times in 12% trichloroacetic acid, then resuspended in 5% SDS. and counted.

SDS-PAGE and lmmunoblottlng Samples for immunoprecipitalion were prepared as in Sztul et al. (1963). Protein samples were subjected to SDS-PAGE as previously described (Sztul et al., 1983) except that 0.8 mm gels were used. Gels were processed for fluorography with En3Hance (NEN, Boston, MA). When indicated, lanes were scanned and the peaks integrated with a scanning densitometer. Upon completion of electrophoresis, the separated proteins were transferred to nitrocellulose filters (Schleicher and Schuell, Keene, NH) at a constant current (150 mA) for at least 4 hr at room temperature. The filters were quenched in 5% dry milk solution (1 hr at room temperature) and then incubated with the primary antibody in the same solution. The filters were washed (twice for 10 min) with PBS, then with PBS containing 0.05% Nonidet P-40 (twice for 10 min), and finally rinsed (twice for 5 min) with PBS. Adsorbed IgGs were detected by incubating the filters with a secondary goat anti-rabbit antibody conjugated to alkaline phosphatase. The detecting antibody was used at a I:5000 dilution in 5% dry milk solution. The alkaline phosphatase reaction was carried out according to the manufacturer’s directions.

Immunoleolatlon Protein A-Sepharose beads (Pharmacia Fine Chemicals, Piscataway, NJ) were incubated overnight at 4% with mouse IgGs against the cytoplasmic domain of p&&R. The immunoadsorbent was washed with PBS. For the immunoisolation, the crude vesicular fraction and the immunoadsorbent were incubated (with mixing) overnight at 4%. Following immunoisolation, the immunoadsorbent with any attached material was retrieved by centrifugation (1600 rpm for 1 min) and washed with 0.25 M sucrose. The starting material (Le., crude vesicular fraction), the

nonbound material, and the material bound to the immunoadsorbent were subjected to SDS-PAGE and immunoblotting with the various antibodies.

Acknowledgments We wish to thank Dr. J.-P Kraehenbuhl for providing us with the myeloma cell line producing anti-pIgA-R antibodies. We are grateful to Drs. Ann Hubbard, David Bole, Robert Levenson, and John Woods for their kind gifts of antibodies. We thank Kathryn Howell for her advice during the course of this work. This research was supported by a grant from the National Institutes of Health (GM 27303 to G. E. P) and by a pilot project from the Yale University School of Medicine, Liver Center Dk 34969 (to E. S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘bdvertisement” in accordance with 16 USC Section 1734 solely to indicate this fact. Received

July 16, 1990; revised September

25, 1990.

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