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Vol. 18, Issue 7, 2707-2715, July 2007
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*Department of Biology, The Catholic University of America, Washington, DC 20064; and Graduate Environmental Studies Unit, The Evergreen State College, Olympia, WA 98505
Submitted February 2, 2007;
Revised April 2, 2007;
Accepted May 1, 2007
Monitoring Editor: Robert Parton
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unlike most simple epithelial cells, hepatocytes use the indirect pathway for apical delivery of newly synthesized single transmembrane domain (TMD) and glycophosphatidylinositol (GPI)-anchored proteins (Bartles et al., 1987
; Bartles and Hubbard, 1988; Schell et al., 1992; Bastaki et al., 2002; Tuma and Hubbard, 2003). We have previously shown that transcytotic sorting from the early endosome to the subapical compartment (SAC) in WIF-B cells requires cholesterol and glycosphingolipids (Nyasae et al., 2003). In cells depleted of these lipids, efflux from early endosome of all apical residents examined and polymeric IgA receptor (pIgA-R) was significantly impaired irrespective of their detergent solubility properties. This indicated that apical residents do not require direct lipid-association for transcytotic sorting. Rather, the lipid-dependent sorting was likely conferred by a general regulator of transcytosis whose activity requires cholesterol and glycosphingolipids. Similarly, cholesterol depletion in Madin-Darby canine kidney (MDCK) cells impaired the direct apical transport of different classes of apical residents and secretory proteins irrespective of their solubility properties (Scheiffele et al., 1997; Keller and Simons, 1998; Prydz and Simons, 2001). Thus, lipid-dependent sorting at the TGN is likely regulated by a general, lipid-associated molecule.
The myelin and lymphocyte protein (MAL) proteolipids are good candidates for mediating lipid-dependent apical sorting. These 
20-kDa tetraspanning membrane proteins are raft associated, and they have been implicated as important regulators of apical delivery in both the direct and indirect pathways. In MDCK cells lacking MAL, direct apical delivery was decreased; the ectopic expression of MAL rescued the defect (Cheong et al., 1999; Puertollano et al., 1999, 2001; Martin-Belmonte et al., 2000, 2001). Because apical secretion of different classes of secretory proteins (thyroglobulin and gp80) and apical delivery of different classes of apical residents (single TMD and GPI anchored) were both impaired in MAL-depleted cells, we consider MAL a general regulator of direct apical transport. Although MDCK cells express MAL2, its role in transcytosis remains unclear (Wilson et al., 2001; De Marco et al., 2002) (see Discussion).
In contrast, hepatocytes express only MAL2 (Alonso and Weissman, 1987; Wilson et al., 2001; De Marco et al., 2002), consistent with the absence of direct apical delivery of single TMD and GPI-anchored residents. In HepG2 cells, antisense MAL2 oligonucleotides impaired transcytosis of two classes of apical proteins: pIgA via its single TMD receptor and CD59, a GPI-anchored protein (De Marco et al., 2002). Interestingly, the block occurred between early endosomes and the SAC, reminiscent of the transcytosis defect we observed in lipid-depleted WIF-B cells (Nyasae et al., 2003). Thus, lipid depletion may prevent MAL2 from sorting transcytosing proteins at early endosomes and prevent MAL from sorting at the TGN.
Does MAL expression reroute newly synthesized apical proteins into the direct pathway in hepatocytes? To answer this question, we expressed MAL in polarized, hepatic WIF-B cells and examined the distributions of different classes of apical proteins. We found that MAL expression induced the formation cholesterol and glycosphingolipid-enriched Golgi domains that contained GPI-anchored and single TMD apical residents; polytopic apical proteins, basolateral residents, and pIgA-R were excluded. Basolateral surface labeling revealed decreased basolateral delivery of GPI-anchored and single TMD apical residents in MAL-expressing cells, whereas basolateral amounts of pIgA-R and basolateral residents were unchanged. By using a quantitative morphological assay, we determined that MAL was rerouting apical proteins into a direct pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cyclodextrin (mCD) were purchased from Sigma-Aldrich (St. Louis, MO). CHX was made fresh in 5% ethanol, and mCD was made fresh in serum-free medium. BFA was stored at –20°C as a 10 mg/ml stock in dimethyl sulfoxide. Horseradish peroxidase (HRP)-conjugated secondary antibodies and Super Signal West Pico enhanced chemiluminescence (ECL) substrate were from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) and Pierce Chemical (Rockford, IL), respectively. Alexa-conjugated secondary antibodies were from Invitrogen (Carlsbad, CA). Anti-myc epitope tag antibodies and anti-MAL polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-5'nucleotidase (5'NT) (monoclonal and affinity purified polyclonal), anti-hemagglutinin (HA), and anti-multidrug resistance-associated protein 2 (MRP2) antibodies were kindly provided by J. P. Luzio (Cambridge University, Cambridge, United Kingdom) M. Roth (University of Texas, Southwestern, Dallas, TX), and D. Keppler (Deutsches Krebsforschungszentrum, Heidelberg, Germany), respectively. Antibodies against aminopeptidase N (APN), CE9, pIgA-R, dipeptidyl peptidase IV (DPP IV), HA321, and myc epitope tag (9E10) were all generously provided by A. Hubbard (Johns Hopkins University, School of Medicine, Baltimore, MD). Recombinant adenoviruses encoding V5/His6 epitope-tagged full-length DPP IV or pIgA-R and full-length HA were also all provided by A. Hubbard, and they have been described in detail previously (Bastaki et al., 2002). A cDNA encoding full-length MAL was kindly provided by M. Alonso (Severo Ochoa Center for Molecular Biology, Universidad Autonoma de Madrid, Madrid, Spain).
Cell Culture, Virus Production, and Infection
WIF-B cells were grown in a humidified 7% CO2 incubator at 37°C as described previously (Shanks et al., 1994). Briefly, cells were grown in F12 medium (Coon modification), pH 7.0, supplemented with 5% fetal bovine serum, 10 µM hypoxanthine, 40 nM aminopterin, and 1.6 µM thymidine. In general, cells were seeded onto glass coverslips at 1.3 x 104 cells/cm2 and grown for 8–12 d until they reached maximum density and polarity.
Recombinant MAL-myc adenoviruses were generated using the Cre-Lox system as described previously (Bastaki et al., 2002). WIF-B cells were infected with recombinant adenovirus particles (0.7–1.4 x 1010 virus particles/ml) encoding myc-epitope tagged MAL, green fluorescent protein (GFP), or V5/His6 epitope-tagged DPP IV or pIgA-R or untagged HA for 60 min at 37°C as described previously (Bastaki et al., 2002). The cells were washed with complete medium and incubated an additional 18–20 h to allow expression.
Immunofluorescence Microscopy
In general, cells were fixed on ice with chilled phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 1 min and permeabilized with ice-cold methanol for 10 min. To detect MRP2, cells were fixed and permeabilized at –20°C with methanol for 5 min. To detect MAL with anti-MAL antibodies, cells were fixed for 30 min with 4% PFA at room temperature (RT) and permeabilized for 10 min at RT with 0.2% Triton X-100/PBS. Cells were processed for indirect immunofluorescence as described previously (Ihrke et al., 1993). Alexa 488- or 568-conjugated secondary antibodies were used at 5 µg/ml. For some experiments, cells were treated for 1 h with 10 µg/ml BFA or up to 2 h with 50 µg/ml CHX at 37°C. To deplete cholesterol, cells were treated for 1 h with 1 or 5 mM mCD in serum-free medium.
Labeled cells were visualized by epifluorescence on an Olympus BX60 fluorescence microscope (Opelco, Dulles, VA). Images were taken using an HQ2 digital camera (Photometrics, Tucson, AZ) and IPLabs image analysis software (Biovision, Exton, PA) or by using a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI) and SPOT Advanced software, version 3.5.8 (Diagnostic Instruments). Adobe Photoshop (Adobe Systems, Mountain View, CA) was used to compile figures.
Cells expressing HA, pIgA-R, or DPP IV alone or with MAL were treated for increasing times with 50 µg/ml CHX. Then, cells were fixed, permeabilized, and stained. Random fields were visualized by epifluorescence and digitized. From micrographs, the average pixel intensity of selected regions of interest (ROI) placed at the apical or basolateral PM of the same WIF-B cell were measured using the Measure ROI tool of the ImageJ imaging software (National Institutes of Health, Bethesda, MD). In general, multiple ROI were collected in the same cell to verify that representative intensities were measured. The averaged background pixel intensity was subtracted from each value, and the ratio of apical-to-basolateral PM fluorescence intensity was determined. Approximately 100–300 cells were measured for each condition from at least three independent experiments. Values are expressed as the mean ± SEM.
Low Buoyant Density Flotations
Isolation of low buoyancy membrane fractions was performed as described previously (Brown and Rose, 1992). Control or MAL-infected WIF-B cells were rinsed in cold PBS and lysed on ice for 30 min with ice-cold lysis buffer (1% [vol/vol] Triton X-100, 150 mM NaCl, and 5 mM EDTA, pH 7.4) containing 1 µg/ml each of antipain, leupeptin, benzamidine, and phenylmethylsulfonyl fluoride. The lysates were diluted with an equal volume of lysis buffer containing 80% sucrose and placed in the bottom of a 12-ml centrifuge tube. A 5–30% linear sucrose gradient was poured on top of the extracts, and the tubes were centrifuged in a swinging bucket rotor at 192,000 x g for 16 h at 4°C. One-milliliter fractions were collected from the bottom and immunoblotted with the indicated antibodies. HRP-conjugated secondary antibodies were used and immunoreactivity was detected with ECL. The relative distributions of the different proteins were determined by densitometric comparison of immunoreactive bands.
Antibody Labeling of Live Cells
Cells were cooled on ice for 5 min at 4°C. Selected PM proteins were surface labeled with specific antibodies for 20 min at 4°C. Because tight junctions restricted access of the antibodies to the apical PM, only antigens at the basolateral surface were labeled. For transcytosis assays, cells were washed two times for 2 min on ice and reincubated with prewarmed complete medium. Antibodies with bound antigens were allowed to chase for the indicated times at 37°C, and cells were fixed and stained.
For basolateral surface-labeling experiments, after antibody labeling on ice, cells were lysed by addition of SDS-polyacrylamide gel electrophoresis sample buffer. Lysates were immunoblotted with the indicated primary antibodies to detect the entire population of the selected PM protein. On a parallel immunoblot, lysates were probed directly with secondary antibodies to detect only the surface bound primary antibodies. The relative levels of immunoreactive species were determined by densitometry. The amount of surface-bound antibodies in control and MAL-infected cells was normalized to the amount of total antigen present. In all cases, control ratios were set to 100%.
Internalization Assays
Total IgG from serum (DPP IV) or ascites (5'NT) was purified (EZ-Sep; Pharmacia AB, Uppsala, Sweden) and biotinylated (EZ-Link sulfo-NHS-biotin; Pierce Chemical) according to the manufacturers' instructions. Internalization assays were performed as described previously (Tuma et al., 2002). Briefly, WIF-B cells were continuously labeled with biotinylated antibodies for the indicated times at 37°C. The remaining surface-associated antibodies were eluted with isoglycine (200 mM glycine and 150 mM NaCl, pH 2.5) for 5 min at RT, and the cells lysed in isoglycine containing 20 mM octylglucoside and 0.5% Triton X-100 for 30 min on ice. Aliquots of the eluate and lysate were incubated in streptavidin-coated 96-well plates (Pierce Chemical). Bound antibodies were detected with HRP-conjugated secondary antibodies followed by colorimetric detection with an HRP substrate detection kit (Pierce Chemical).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70%; Table 1), which likely reflects lower rates of endogenous protein synthesis.
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The Structures Are Cholesterol and Glycosphingolipid-enriched Golgi Domains
According to the "raft hypothesis" for protein sorting, cholesterol- and glycosphingolipid-enriched domains form in the biosynthetic pathway where they recruit apically destined proteins, and then the rafts and their recruited cargo are transported in vesicles directly to the apical domain (Simons and Ikonen, 1997). Because MAL has been shown to be raft associated and to function at the TGN, we tested whether MAL expression induced the formation of biosynthetic, cholesterol- and glycosphingolipid-enriched Golgi domains in WIF-B cells. We first examined whether HA in MAL-expressing cells was present in the Golgi. In control cells, HA was primarily localized to the apical PM, showing no overlap with the Golgi marker albumin (Figure 2A, a–c). In contrast, the intracellular HA in MAL-overexpressing cells largely colocalized with albumin (Figure 2A, c–f), indicating its presence in the Golgi.
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If MAL expression induced biosynthetic raft formation, one prediction was that cholesterol depletion should impair accumulation of the apical proteins in the Golgi domains. To test this, we treated cells with mCD for 60 min, conditions that deplete 80% of cholesterol in WIF-B cells (Nyasae et al., 2003). However, in 85% of cells expressing MAL, HA remained in the Golgi in treated cells (Figure 3Ab). Because direct targeting requires cholesterol (Scheiffele et al., 1997; Keller and Simons, 1998; Prydz and Simons, 2001), another possibility was that apical staining should persist in cholesterol-depleted cells reflecting impaired apical delivery. To test this prediction, we incubated CHX-treated cells with mCD, conditions whereby "chase" from the compartment could be monitored. As shown above, CHX treatment decreased intracellular HA staining, indicating that apical delivery occurred (Figure 3Bb). However, in cells also treated with mCD, the intracellular HA staining remained (Figure 3Bc and Table 1), indicating that apical delivery was impaired and thus cholesterol dependent.
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, 2000; Tall et al., 2003). As predicted, the raft association of the apical residents was altered by MAL expression. All of APN and 46% of exogenously expressed DPP IV were found in low-density fractions (Figure 4A). Only the high-molecular-weight mature forms of DPP IV were found in the floated fractions, consistent with the identification of the MAL-induced structures as Golgi derived. 5'NT distributions also changed in cells expressing MAL, and no longer distributed to the top of the gradient (Figure 4B). Although 5'NT levels peaked in fractions 5–7 that were less dense than the peak of MAL in fractions 4–6, the two molecules significantly overlapped. The shift down in density for APN and DPP IV, and the shift up for 5'NT into fractions containing MAL, suggest that MAL was enhancing raft association of the apical residents. For comparison, the basolateral resident CE9 remained in the load fraction in preparations from MAL-expressing cells (Figure 4B). Similarly, pIgA-R remained in the load fractions in control and MAL-expressing cells (>97 and 100%, respectively), consistent with the lack of pIgA-R redistribution (Figure 4C). Also importantly, MAL2 distributions were not changed in MAL-infected cells (unpublished data).
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As predicted, the basolateral populations of HA, DPP IV and APN were significantly decreased in MAL-expressing cells (Figure 5A). HA and APN basolateral labeling was decreased by 80% whereas DPP IV labeling was reduced by 50%. The basolateral population of 5'NT was decreased to a lesser extent (70% of control) which is consistent with the fewer number of MAL-expressing cells with intracellular 5'NT staining (Table 1). In contrast, no significant decrease in labeling was observed for basolateral residents (HA321 and CE9) or for pIgA-R (Figure 5A). Greater than 90% of control amounts were detected at the basolateral PM in MAL-infected cells. These results suggest that the basolateral delivery of single TMD and GPI-anchored apical residents was reduced in MAL-expressing cells, while delivery of basolateral residents and pIgA-R was not changed.
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To rule out that any of the intracellular HA in MAL-infected cells was present on transcytotic intermediates, we performed two sets of experiments. First, we colabeled steady-state HA with transcytosing APN. Although less APN was present at the basolateral surface, sufficient labeling was achieved to monitor its apical delivery. After 45 min of chase, APN was present at the apical PM, indicating that transcytosis was not impaired in MAL-expressing cells (Figure 6Ab). Trafficked APN was also detected in small puncta (Figure 6Ab) in MAL-expressing cells, but these puncta did not overlap with the intracellular HA (Figure 6Ac), indicating that HA was not in transcytotic intermediates, confirming its Golgi localization.
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To discriminate between retention versus redirection of apical proteins, we developed a morphological "pulse-chase" analysis where we measured relative fluorescence intensities of a cohort of HA, DPP IV, or pIgA-R at the apical or basolateral PM in CHX-treated cells. We first examined the transcytotic delivery of HA, DPP IV, and pIgA-R in control cells to determine the feasibility of our assay. As shown in Figure 7, the ratio of apical-to-basolateral fluorescence decreased for all markers after 15 min of CHX treatment (75% of 0 min for HA and DPP IV and 85% for pIgA-R), indicating that each cohort was being delivered to the basolateral PM (a decreased ratio reflects increased basolateral delivery). For DPP IV, peak basolateral delivery was seen after 30 min, indicating it was delivered more slowly. After 60 min, the ratios for all three proteins increased beyond that observed at 0 min, signifying that the proteins had traversed the basolateral PM. The ratios for pIgA-R increased much more rapidly (150% vs. 95%), indicating the receptor was more rapidly internalized and transcytosed. By 120 min, all three apical proteins had achieved ratios >100%, indicating successful apical delivery confirming our assay was monitoring transcytosis.
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170%, indicating increased apical delivery. Thus, we conclude that HA and DPP IV were directly delivered to the apical PM from the Golgi. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our Working Model
MAL is mainly expressed at the apical PM, yet its sorting activity is thought to occur at the TGN, whereas MAL2 is found at the SAC in hepatic cells, yet it is thought to sort transcytosing residents from early endosomes. Thus, the MAL proteolipids must be itinerant proteins, a conclusion consistent with findings from Cos7 cells and live cell imaging (Puertollano and Alonso, 1999; de Marco et al., 2006). In our model, which is based on and similar to those previously proposed (e.g., Martin-Belmonte et al., 2001; Puertollano et al., 2001; de Marco et al., 2006), MAL normally encounters apical residents in cholesterol and glycosphingolipid-enriched domains in the TGN, whereas MAL2 encounters apical proteins in similar domains at early endosomes. The proteolipids and newly synthesized apical proteins associate; are packaged into vesicles; and the divergent, cytoplasmic N-terminal domains of each MAL isoform recruit specific (yet unidentified) regulators that target vesicles to either the apical PM or SAC. Because hepatocytes lack MAL, apical sorting at the TGN does not occur, and apical proteins are routed instead into the transcytotic pathway where MAL2-mediated sorting occurs at the basolateral early endosome. Thus, in MAL-expressing hepatic cells, we propose that the newly synthesized apical proteins encountered MAL first at the TGN, and this association rerouted them directly to the apical PM, bypassing interactions with MAL2 and the transcytotic pathway.
This hypothesis is consistent with studies from MDCK cells where apical residents were missorted to the basolateral PM when MAL expression was knocked down (Cheong et al., 1999; Puertollano et al., 1999, 2001; Martin-Belmonte et al., 2000, 2001). Curiously, despite the endogenous expression of MAL2 in MDCK cells, the missorted apical proteins were not transcytosed to the apical PM. One possibility is that MAL2 was not expressed at high enough levels to handle the large load of missorted proteins. Alternatively, MAL2 may regulate transcytotic sorting of different cargo. Thus, it remains to be determined what the specific role of MAL2 is in MDCK cells. Also in MDCK cells, MAL has been implicated in regulating apical endocytosis (Puertollano et al., 2001; Martin-Belmonte et al., 2003). Whether MAL functioned similarly in WIF-B cells remains to be determined.
Intestinal cells express both MAL and MAL2 (De Marco et al., 2002; Marazuela et al., 2003, 2004; Marazuela and Alonso, 2004). If our model is correct, the prediction is that MAL should directly target proteins to the apical membrane. However, intestinal cells rely on both the indirect and the direct pathways for delivery of newly synthesized apical proteins (Tuma and Hubbard, 2003). In our studies, exogenous MAL expression did not fully reroute the apical proteins (Figure 5A), suggesting that relative levels of MAL and the newly synthesized apical proteins are important, i.e., high levels of MAL expression are required for efficient direct targeting. Studies are needed to first confirm the roles of MAL and MAL2 in intestinal apical delivery and second to examine the rates of apical protein synthesis and delivery routes with respect to MAL and MAL2 expression levels.
MAL Reroutes a Subset of Resident Apical Proteins
Exogenous MAL altered the apical delivery of single TMD and GPI-anchored apical proteins, whereas trafficking of pIgA-R, basolateral residents, and the polytopic apical resident MRP2 was not affected. How did the latter three classes of proteins elude MAL-mediated sorting? The cytoplasmic tails of pIgA-R and basolateral residents encode targeting signals that mediate their delivery to the basolateral PM (Casanova et al., 1991; Keller and Simons, 1997). We propose that these targeting signals are dominant or independent to the sorting provided by MAL. This explains why pIgA-R takes the indirect route in MDCK cells where most other single span proteins take the direct pathway. This conclusion is also consistent with the finding that many apical targeting signals can function only in the absence of basolateral targeting signals (Matter and Mellman, 1994; Keller and Simons, 1997; Rodriguez-Boulan et al., 2005). The apical sorting signal for MRP2 has been mapped to its cytoplasmic, C-terminal domain that contains a PDZ-binding motif (Harris et al., 2001; Nies et al., 2002). Similarly, other apical multispanning proteins may be sorted to the apical PM via these motifs (Fanning and Anderson, 1999). We suggest, that like the basolateral targeting signals, these motifs are dominant to the sorting conferred by MAL.
In contrast, DPP IV, APN, and HA encode short, cytoplasmic tails (6, 8, and 12 amino acids, respectively) that contain no known targeting information. Because of the shortness of these cytoplasmic domains and the lack of cytoplasmic regions of GPI-anchored proteins, we suggest that sorting is not directly conferred via cytosolic targeting proteins. Rather, we favor the possibility that exogenous MAL redirected these (and other?) apical proteins via interactions that occurred within the bilayer. Consistent with this hypothesis is the finding that the 10 amino acids of the TMD of HA that span the bilayer outer leaflet were important for apical targeting, raft association, and MAL binding (Lin et al., 1998; Tall et al., 2003). Within this 10 amino acids, glycine 520 and serine 521 have been shown to mediate apical delivery (Scheiffele et al., 1997; Lin et al., 1998) which are not in the TMDs of APN or DPP IV. In fact, there is virtually no sequence conservation between the HA TMD and those of DPP IV or APN. This lack of sequence conservation suggests that the interactions between MAL and the apical proteins are indirect or weak. This is consistent with our inability to coimmunoprecipitate MAL with any apical protein (data not shown). Similarly, only 0.7–2% of HA was coimmunoprecipitated with MAL in MDCK cells (Tall et al., 2003).
Interestingly, each of the TMD sequences of the MAL family members share significant sequence identities (Sanchez-Pulido et al., 2002), suggesting that these domains regulate specific interactions with proteins or lipids that might be required for sorting. Recently, it was proposed that oligomerization of GPI-anchored proteins promotes raft association that then mediates apical delivery (Paladino et al., 2004). The authors suggest that the association is driven either by oligomerization and subsequent stabilization into rafts or by coalescence of rafts that then promote oligomerization. One possibility is that MAL promotes raft formation or clustering that is required for apical sorting, a hypothesis we are actively examining.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Pamela L. Tuma (tuma{at}cua.edu)
Abbreviations used: 5'NT, 5'nucleotidase; APN, aminopeptidase N; BC, bile canaliculus; BFA, brefeldin A; CHX, cycloheximide; DPP IV, dipeptidyl peptidase IV; GPI, glycophosphatidylinositol; HA, hemagglutinin; MAL, myelin and lymphocyte protein; mCD, methyl--cyclodextrin; MRP2, multidrug resistance-associated protein 2; pIgA-R, polymeric IgA receptor; PM, plasma membrane; SAC, subapical compartment; TMD, transmembrane domain.
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REFERENCES |
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