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The H,K-ATPase β Subunit as a Model to Study the Role of N-Glycosylation in Membrane Trafficking and Apical Sorting

2004; Elsevier BV; Volume: 279; Issue: 37 Linguagem: Inglês

10.1074/jbc.m405453200

1083-351X

Olga Vagin, Shahlo Turdikulova, George Sachs,

Ion Transport and Channel Regulation

The role of N-glycosylation in trafficking of an apical membrane protein, the gastric H,K-ATPase β subunit linked to yellow fluorescent protein, was analyzed in polarized LLC-PK1 cells by confocal microscopy and surface-specific biotinylation. Deletion of the N-glycosylation sites at N1, N3, N5, and N7 but not at N2, N4, and N6 significantly slowed endoplasmic reticulum-to-Golgi trafficking, impaired apical sorting, and enhanced endocytosis from the apical membrane, resulting in decreased apical expression. Golgi mannosidase inhibition to prevent carbohydrate chain branching and elongation resulted in faster internalization and degradation of the β subunit, indicating that terminal glycosylation is important for stabilization of the protein in the apical membrane and protection of internalized protein from targeting to the degradation pathway. The decrease in the apical content of the β subunit was less with mannosidase inhibition compared with that found in the N1, N3, N5, and N7 site mutants, suggesting that the core region sugars are more important than the terminal sugars for apical sorting. The role of N-glycosylation in trafficking of an apical membrane protein, the gastric H,K-ATPase β subunit linked to yellow fluorescent protein, was analyzed in polarized LLC-PK1 cells by confocal microscopy and surface-specific biotinylation. Deletion of the N-glycosylation sites at N1, N3, N5, and N7 but not at N2, N4, and N6 significantly slowed endoplasmic reticulum-to-Golgi trafficking, impaired apical sorting, and enhanced endocytosis from the apical membrane, resulting in decreased apical expression. Golgi mannosidase inhibition to prevent carbohydrate chain branching and elongation resulted in faster internalization and degradation of the β subunit, indicating that terminal glycosylation is important for stabilization of the protein in the apical membrane and protection of internalized protein from targeting to the degradation pathway. The decrease in the apical content of the β subunit was less with mannosidase inhibition compared with that found in the N1, N3, N5, and N7 site mutants, suggesting that the core region sugars are more important than the terminal sugars for apical sorting. Sorting of proteins between apical and basolateral membranes in polarized cells depends on the recognition of intrinsic sorting signals within the proteins by specific sorting machinery in the trans-Golgi network (TGN) 1The abbreviations used are: TGN, trans-Golgi network; ER, endoplasmic reticulum; YFP, yellow fluorescent protein; dMAN, deoxymannojirimycin; PNGase F, peptide N-glycosidase F; Endo H, endoglycosidase H. or endosomes (1Traub L.M. Kornfeld S. Curr. Opin. Cell Biol. 1997; 9: 527-533Crossref PubMed Scopus (194) Google Scholar, 2Ikonen E. Simons K. Semin. Cell Dev. Biol. 1998; 9: 503-509Crossref PubMed Scopus (152) Google Scholar, 3Yeaman C. Grindstaff K.K. Nelson W.J. Physiol. Rev. 1999; 79: 73-98Crossref PubMed Scopus (444) Google Scholar, 4Nelson W.J. Rodriguez-Boulan E. Nat. Cell Biol. 2004; 6: 282-284Crossref PubMed Scopus (16) Google Scholar). Basolateral sorting signals often contain tyrosine-based, dileucine, or other hydrophobic motifs that are recognized by clathrin coat proteins that package them into basolateral transport vesicles (5Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (393) Google Scholar, 6Keller P. Simons K. J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar). Apical sorting signals are less well defined. N-Linked glycans are considered as possible candidates based on the findings that some proteins gain the ability to reach the apical membrane after recombinant addition of N-glycosylation sites (7Gut A. Kappeler F. Hyka N. Balda M.S. Hauri H.P. Matter K. EMBO J. 1998; 17: 1919-1929Crossref PubMed Scopus (175) Google Scholar) and that other proteins entirely or partially lose apical expression as a result of removal of N-glycans by mutation or treatment with glycosylation inhibitors (5Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (393) Google Scholar, 8Scheiffele P. Peranen J. Simons K. Nature. 1995; 378: 96-98Crossref PubMed Scopus (417) Google Scholar, 9Potter B.A. Ihrke G. Bruns J.R. Weixel K.M. Weisz O.A. Mol. Biol. Cell. 2004; 15: 1407-1416Crossref PubMed Scopus (48) Google Scholar, 10Hendriks G. Koudijs M. van Balkom B.W. Oorschot V. Klumperman J. Deen P.M. van der Sluijs P. J. Biol. Chem. 2004; 279: 2975-2983Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). N-Glycosylation might significantly affect other trafficking steps, including protein folding and quality control in the endoplasmic reticulum (ER), endocytosis, recycling, and degradation. Because the effect of N-glycan removal on an apical sorting event has not been differentiated from a possible impairment of other trafficking steps, the role of N-glycans as apical sorting signals remains unclear. An interesting paradigm for the study of the role of glycosylation in protein maturation, trafficking, sorting, and degradation is an apically targeted multiply glycosylated protein, the H,K-ATPase β subunit. The gastric H,K-ATPase, the enzyme responsible for acid secretion in the stomach, consists of two subunits, a catalytic α subunit and an accessory β subunit, which has seven N-glycosylation sites. Glycosylation of the β subunit has been shown to be critical for the quality control of the H,K-ATPases in the ER (11Geering K. J. Bioenerg. Biomembr. 2001; 33: 425-438Crossref PubMed Scopus (270) Google Scholar, 12Asano S. Kawada K. Kimura T. Grishin A.V. Caplan M.J. Takeguchi N. J. Biol. Chem. 2000; 275: 8324-8330Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Specific N-glycosylation sites have been found to be essential for plasma membrane delivery of the H,K-ATPase β subunit in non-polarized HEK-293 (13Vagin O. Denevich S. Sachs G. Am. J. Physiol. 2003; 285: C968-C976Crossref Scopus (23) Google Scholar) and for delivery of both α and β subunits in COS-7 cells (12Asano S. Kawada K. Kimura T. Grishin A.V. Caplan M.J. Takeguchi N. J. Biol. Chem. 2000; 275: 8324-8330Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), suggesting that they may also play a role in apical sorting. The homologous Na,K-ATPase also consists of α and β subunits. Of four known isoforms of the Na,K-ATPase β subunits, β2 is the most homologous to the H,K-ATPase β subunit, has up to nine glycosylation sites (14Blanco G. Mercer R.W. Am. J. Physiol. 1998; 275: F633-F650PubMed Google Scholar), and appears to result in apical sorting of the Na,K-ATPase α·β complex in a number of tissues (15Wilson P.D. N. Engl. J. Med. 2004; 350: 151-164Crossref PubMed Scopus (617) Google Scholar, 16Wilson P.D. Devuyst O. Li X. Gatti L. Falkenstein D. Robinson S. Fambrough D. Burrow C.R. Am. J. Pathol. 2000; 156: 253-268Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 17Mobasheri A. Oukrif D. Dawodu S.P. Sinha M. Greenwell P. Stewart D. Djamgoz M.B. Foster C.S. Martin-Vasallo P. Mobasheri R. Histol. Histopathol. 2001; 16: 141-154PubMed Google Scholar). The Na,K-ATPase that contains either the β1 or β3 isoform with only two or three N-glycosylation sites localizes exclusively in the basolateral membrane (18Caplan M.J. Am. J. Physiol. 1997; 272: G1304-G1313PubMed Google Scholar). The high degree of glycosylation in the H,K-ATPase β and Na,K-ATPase β2 subunits might imply a role of N-glycosylation in the apical sorting of the corresponding α·β complexes. N-Glycosylation occurs in various stages. N-Linked oligosaccharides appear when the 14-saccharide core is transferred from the dolichol phosphate precursor to the nascent membrane protein that remains associated with the translocon in the ER. Immediately after coupling the core to the asparagine of the specific amino acid NXS or NXT motif, the N-glycosylation site, the terminal glucose residues are trimmed by ER glucosidases. Various chaperones such as calnexin bind to sugar chains as quality control elements at this and perhaps later stages. Subsequently, the mannose residues are trimmed in the ER and then in the Golgi by mannosidases I and II, respectively. This is followed by elongation of the carbohydrate chains due to addition of terminal sugars by the action of various glycosyltransferases in the trans-Golgi (19Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1984) Google Scholar). There is considerable variation in the number and composition of terminal chains in the mature complex oligosaccharides, giving rise to heterogeneity of glycosylated proteins. The core region that contains five residues originating from the initial 14-saccharide core is the same in all molecular species of mature glycoproteins. Deletion of any N-glycosylation site in a protein abrogates the whole oligosaccharide tree at that particular locus, leaving the others intact. Inhibition of glucose or mannose trimming prevents addition of the terminal sugar chains but leaves core regions intact at all N-glycosylation sites. This enables distinctions to be made concerning the roles of core region and terminal sugars at various stages of processing and trafficking of the protein. Here, we quantitatively determined at which trafficking stages N-glycosylation deficiency affects apical polarity. We determined the effect of either deletion of individual N-glycosylation sites or treatment of the cells with N-glycan-trimming inhibitors on the steady-state distribution of the H,K-ATPase β subunit between the ER, Golgi, and apical and basolateral membranes as well as on endocytosis efficiency and degradation rate in polarized LLC-PK1 cells by quantifying the immature and mature forms of the protein that differ in their molecular masses and by using surface-specific biotinylation and confocal microscopy. Glycosylation at the N1, N3, N5, and N7 sites of the gastric H,K-ATPase β subunit is crucial for ER-to-Golgi trafficking, apical membrane delivery, and stability of the protein in the apical membrane. Considering the steady-state apical content of the mature H,K-ATPase β subunit as a result of dynamic equilibrium between two opposite processes, the apical membrane delivery from TGN/endosomes, and endocytosis from the apical membrane, we were able to quantify the contribution of N-glycosylation to each of these steps. In addition, we show that particular carbohydrate residues of the N-glycans have different roles in apical membrane delivery and endocytosis, which is the first indication for a specific role of certain carbohydrates in apical sorting. In the mature complex-type glycoprotein, the core region sugars are more important for the apical sorting event in TGN/endosomes compared with terminal sugars, whereas the terminal sugars anchor the protein on the apical membrane and also protect the protein from targeting to the degradation pathway after endocytosis. Construction of cDNAs Encoding Yellow Fluorescent Protein (YFP)H,K-ATPase β Subunit Fusion Proteins and Mutants Lacking Glycosylation Sites—pcDNA3(+)β (20Lambrecht N. Munson K. Vagin O. Sachs G. J. Biol. Chem. 2000; 275: 4041-4048Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) was used as a source for cDNA encoding the rabbit H,K-ATPase β subunit (GenBank™/EBI accession number M35544) (21Reuben M.A. Lasater L.S. Sachs G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6767-6771Crossref PubMed Scopus (138) Google Scholar). The cDNA encoding the β subunit was inserted into the multiple cloning site of the expression vector pEYFP-C1 (Clontech) using BglII and BamHI restriction sites to form pEYFP-β, which encodes YFP-β, a fusion protein of YFP linked to the N terminus of the H,K-ATPase β subunit. Mutants were generated using the QuikChange mutagenesis kit (Stratagene). The rabbit gastric H,K-ATPase β subunit has seven N-glycosylation sites in the extracellular C terminus. Seven single mutants of YFP-β fusion protein lacking a single N-glycosylation site, N99Q (N1), N103Q (N2), N130Q (N3), N146Q (N4), N161Q (N5), N193Q (N6), and N222Q (N7), were constructed using pEYFP-β as a template. Stable Transfection—To obtain cell lines stably expressing wild-type or mutant YFP-β fusion proteins, LLC-PK1 cells were grown on 10-cm plates until 20% confluent and transfected with wild-type YFP-β or mutant YFP-β using FuGENE 6 transfection reagent (Roche Applied Science). 24 h after transfection, stable cell lines were selected by addition of the eukaryotic selection marker G418 at a final concentration of 1.0 mg/ml. This concentration of G418 was maintained until single colonies appeared. 15-20 colonies were isolated, expanded, and grown in the presence of 0.25 mg of G418/ml of medium in 24-well plates. Two clones with the highest expression of YFP-β were selected and expanded for further studies. Confocal Microscopy Studies—Cells stably expressing wild-type or mutant YFP-β were grown for at least 5 days after becoming confluent on glass bottom micro-well dishes (Mattek Corp.). Confocal microscopy images were acquired using a Zeiss LSM 510 laser scanning confocal microscope with LSM 510 Version 3.2 software. Estimation of Surface YFP-β Content by Surface-specific Biotinylation—LLC-PK1 cells stably expressing wild-type or mutant YFP-β were maintained for at least 5 days after becoming confluent in Corning Costar polyester Transwell inserts (Corning Inc.) in 6-well plates. Biotinylation of the apical or basolateral membrane proteins was performed by previously described procedures (22Gottardi C.J. Dunbar L.A. Caplan M.J. Am. J. Physiol. 1995; 268: F285-F295Crossref PubMed Google Scholar, 23Kroepfl J.F. Gardinier M.V. J. Neurochem. 2001; 77: 1301-1309Crossref PubMed Scopus (22) Google Scholar). Briefly, cell mono-layers were biotinylated with EZ-Link™ sulfosuccinimidyl-2-(biotinamido)ethyl 1,3′-dithiopropionate (Pierce), which was added from either the apical or basolateral side. After quenching the biotinylation reaction, cells were washed and then lysed by incubation with 200 μl of 0.15 m NaCl in 15 mm Tris (pH 8.0) containing 1% Triton X-100 and 4 mm EGTA. Cell lysates were clarified by centrifugation at 15,000 × g for 10 min. Samples containing 20 μl of supernatant mixed with 15 μl of SDS-containing sample buffer were loaded onto SDS-polyacrylamide gel to determine the total YFP-β content in the supernatant. To precipitate biotinylated proteins, the rest of each supernatant was incubated with 100 μl of streptavidin-agarose beads (Sigma) in a total volume of 800 μl of the lysis buffer for 1 h at 4 °C with continuous rotation. Precipitated complexes were washed three times on the beads, and then proteins were eluted from the beads by incubation in 40 μl of SDS-PAGE sample buffer (4% SDS, 0.05% bromphenol blue, 20% glycerol, and 1% β-mercaptoethanol in 0.1 m Tris (pH 6.8)) for 5 min at 80 °C, separated on SDS-polyacrylamide gel, and analyzed by Western blotting using monoclonal antibody 2B6 against the H,K-ATPase β subunit (MBL, Inc.) or the monoclonal antibody against the Na,K-ATPase β1 subunit (Novus Biologicals) as the primary antibody and anti-mouse IgG conjugated to alkaline phosphatase (Promega) as the secondary antibody according to the manufacturers' instructions. Endocytosis Assay by Apical Surface Biotinylation—Polarized cells stably expressing wild-type or mutant YFP-β were biotinylated from the apical side as described above. Cells were incubated at 18 °C to prevent apical membrane delivery for 20, 60, or 120 min. After that, apical biotin was stripped off by incubation with 50 mm reduced glutathione (Sigma) in 100 mm NaCl with 10% fetal bovine serum (pH 8.4) twice for 20 min. After cell lysis, the internalized biotinylated proteins were precipitated, washed, eluted from streptavidin-agarose beads, and analyzed by SDS-PAGE and Western blot analysis as described above. In the negative control, biotin was stripped off immediately after biotinylation. The initial apical content was determined by lysing cells immediately after biotinylation. In the positive control, cells were incubated at 18 °C for 60 or 120 min to account for any instability of biotinylated protein and then lysed. After cell lysis, biotinylated proteins were precipitated, washed, eluted, and analyzed as described above. For the mutants with very low apical content of YFP-β (N1, N3, and N5), each experimental condition was repeated in three wells, and cell lysates from three identical wells were combined before precipitation on streptavidin-agarose beads. Endocytosis efficiency was calculated as a percentage of apical YFP-β that was internalized for 1 h. Treatment of Cells with Glycosidase Inhibitors—Cells were incubated with castanospermine (Sigma), the Golgi mannosidase I inhibitor deoxymannojirimycin (dMAN) (Sigma), or the Golgi mannosidase II inhibitor swainsonine (Sigma) at a concentration of 1, 2, or 200 μg/ml, respectively, for 48 h prior to apical biotinylation. Glycosidase Cleavage—Where indicated, the total cell lysates or proteins precipitated by streptavidin-agarose beads were treated with peptide N-glycosidase F (PNGase F) from Flavobacterium meningosepticum (New England Biolabs Inc.) or endoglycosidase H (Endo H) from Streptomyces plicatus (Glyco-Prozyme Inc.) according to the manufacturers' instructions. In experiments with live cells, sialidase from Salmonella typhimurium recombinant in Escherichia coli (Glyco-Prozyme Inc.), β1,4-galactosidase from Streptococcus pneumonia (Glyco-Prozyme Inc.), or PNGase F from F. meningosepticum was added to the cell medium from the apical side at a concentration of 1 unit/ml, 66 milliunits/ml, or 7500 New England Biolabs units/ml, respectively, and incubated for 16 h. 1 h prior to completion of the cleavage, cycloheximide (Sigma) at a concentration of 10 μg/ml was added to the medium to inhibit de novo synthesis of YFP-β. Analysis of N-Glycosylation Alteration—The H,K-ATPase β subunit has seven N-glycosylation sites. The mature oligosaccharide linked to each of the seven sites consists of the three-mannosyl core region and terminal chains (Fig. 1). Three approaches were used to analyze the effect of N-glycosylation deficiency in the β subunit. By mutating each of seven N-glycosylation sites and expressing these mutants in polarized cells, we obtained β subunits lacking all core and terminal sugars at only a single N-glycosylation site, with six sites still normally glycosylated. Alternatively, cells expressing the wild-type β subunit were treated with a glucosidase inhibitor (castanospermine), a Golgi mannosidase I inhibitor (dMAN), or a Golgi mannosidase II inhibitor (swainsonine). The inhibitors prevent glucose or mannose trimming and further elongation of all the carbohydrate chains and thus result in expression of the 14-saccharide core and the high mannose- and hybrid-type oligosaccharides, respectively. These steps are illustrated schematically in Fig. 1A. Thus, the glycoprotein formed in the presence of castanospermine or dMAN lacks all the terminal sugars normally present in the complex-type β subunit; and in the presence of swainsonine, the glycoprotein lacks the majority of normal terminal sugars at all seven N-glycosylation sites (Fig. 1A). The complex-, hybrid-, and high mannose-type and 14-saccharide core forms of YFP-β can be clearly distinguished from each other upon SDS-PAGE due to the difference in molecular masses, as shown in Fig. 1B. Characterization of Glycosylated Forms of YFP-β Expressed in LLC-PK1 Cells—YFP-β was detected in cell lysates of LLCPK1 cells as two bands, one at 80-100 kDa and the other at ∼75 kDa (Fig. 2, lane 1). After PNGase F treatment of the cell lysates, the bands at 80-100 and 75 kDa both disappeared, and a single band was seen at ∼55 kDa, corresponding to deglycosylated YFP-β (lane 3). A similar product was detected after treatment of the cell lysates with Endo H, but this treatment resulted in the disappearance of only the lower band on the Western blot, whereas the higher band was retained (lane 2). It is known that Endo H cleaves only high mannose- or hybrid-type glycoproteins, whereas complex-type chains are Endo H-resistant. Therefore, the 80-100-kDa band represents the complex-type glycosylated fraction of YFP-β. The 75-kDa Endo H-sensitive band represents high mannose-type YFP-β (Fig. 1B, fourth lane), as can be concluded from comparison with the lysates prepared from cells preincubated with the glycosylation inhibitors dMAN, which led to formation of only high mannose-type YFP-β (second lane), and swainsonine, which resulted in formation of both hybrid- and high mannose-type glycoproteins (third lane). In contrast to the total cell lysate, the apically biotinylated protein fraction contained only the complex-type glycosylated fraction of YFP-β (Fig. 2, lane 4), which was Endo H-resistant (lane 5). Thus, the mature YFP-β component in LLC-PK1 cells is complex-type glycosylated protein. Only this fraction of the YFP-β pool was able to reach the apical membrane. The high mannose-type core glycosylated fraction of YFP-β present in cell lysates therefore represents the immature ER portion of the YFP-β pool. Surface biotinylation was used to quantify the apical and basolateral content of YFP-β (Fig. 3). Under biotinylation conditions, (i) LLC-PK1 cells may have their tight junctions disrupted during the biotinylation procedure, and (ii) their apical membranes may also become leaky. Hence, in all experiments, biotinylation of basolateral membranes was used to define a specific basolateral location of the Na,K-ATPase β1 subunit (18Caplan M.J. Am. J. Physiol. 1997; 272: G1304-G1313PubMed Google Scholar) as a control for intact tight junctions, and the absence of high mannose-type YFP-β in biotinylated samples was used as an indication of apical membrane integrity. The presence of biotinylated high mannose-type YFP-β protein would show that the biotinylation reagent had access to the intracellular pool of high mannose-type YFP-β because of the apical membrane leakiness during the experiment. As shown in Fig. 3 (left panel), ∼85% of surface YFP-β was detected on the apical membrane. In contrast, the endogenous Na,K-ATPase β1 subunit was predominantly labeled from the basolateral membrane, indicating integrity of tight junctions. Effect of N-Glycosylation Site Mutations on the Relative Apical Content of YFP-β in LLC-PK1 Cells—Using confocal microscopy, the wild-type YFP-β was detected predominantly in the apical region in LLC-PK1 cells (Fig. 4, left panel), as can be seen in the confocal apical XY section of the cell monolayer and particularly in the Z section. A minor fraction of YFP-β was detected inside the cells (XY section through the middle of the cell layer) and in the lateral membranes (Z section). In contrast, the YFP-β mutant lacking the N1 glycosylation site was accumulated mostly intracellularly (right panel). The protein was predominantly localized to the perinuclear region, presumably in the ER and Golgi. To quantify the apical content in the mutants compared with the wild-type protein, cells expressing wild-type or mutant YFP-β were biotinylated from the apical side in the same experiment to prevent variations in biotinylation, streptavidinagarose precipitation, and immunoblotting efficiencies. The apical content was normalized to the total YFP-β content in the corresponding cell lysate for each mutant and compared with that in the wild-type protein, as shown in Fig. 5. The relative apical content was dramatically decreased in N1, N3, and N5 by 14.5-, 7.1-, and 5.5-fold, respectively, and was not detectable in N7. In contrast, mutation of N2, N4, and N6 only moderately decreased the relative apical content (from 1.4- to 1.7-fold). We found that mature YFP-β contains complex-type oligosaccharides only and that the high mannose-type form of the protein corresponds to the ER fraction of the cellular YFP-β pool (Fig. 2). Therefore, the ratio between the complex-type form and total YFP-β can be used as a measure of ER-to-Golgi trafficking. In all the mutants except N6, the relative content of the complex-type glycosylated fraction of YFP-β was decreased, indicating that these mutations slowed down ER-to-Golgi trafficking and caused more ER retention. The most significant effect on ER-to-Golgi trafficking was observed in the N1 and N7 mutants (2.3- and 6.4-fold decreases, respectively). Effect of N-Glycosylation Site Mutations on the Internalization Efficiency of YFP-β in LLC-PK1 Cells—To compare the effect of mutations on the internalization efficiency of YFP-β, the apical surface of cells expressing wild-type or mutant YFP-β was biotinylated, and cells were incubated at 18 °C to prevent apical membrane delivery of internalized proteins. Any apical biotin was then cleaved off, and internalized biotinylated proteins were detected as described under "Experimental Procedures." Internalization efficiency (viz. the fraction of apical YFP-β internalized after 1 h) was increased from 2- to 3-fold in the N1, N3, and N5 mutants, but was only slightly increased in the N1, N4, and N6 mutants (Fig. 6). The internalization efficiency in N7 could not be measured due to the very low apical content of YFP-β in this mutant. Effect of N-Glycosylation Site Mutations on Apical Sorting—The surface distribution of mutants lacking the N3 or N5 site was different from that of the wild-type protein. The major plasma membrane-located fraction of the mutant proteins was detected on the basolateral (but not apical) membrane (Fig. 3). This might imply that removal of the particular N-glycosylation site impaired apical sorting. However, a steady-state distribution between two distinct surface domains is not a result only of apical sorting in the TGN and/or endosomes, but also reflects a balance between apical and basolateral sorting as well as apical and basolateral endocytosis. To quantify the effect of mutations solely on apical sorting, we compared their effect on the relative apical content and the efficiency of endocytosis and calculated the effect of mutations on the apical sorting efficiency. The apical content of YFP-β normalized by comparison with the mature complex-type fraction of YFP-β (CA/CT) (Fig. 5) reflects a steady-state distribution between the apical and internal mature complex-type pools of YFP-β. We found that the rate of degradation of the mature complex-type fraction was not changed by N-glycosylation site mutations. Therefore, the distribution between apical and internal YFP-β could be shifted toward the internal pool in the mutants either because of enhanced endocytosis or because of the impaired apical membrane delivery (see Fig. 10). The relative decrease in the apical content was calculated for each mutant by dividing the apical content in the wild-type protein, (CA/CT)wt, by the apical content in the mutant, (CA/CT)mut (Table I, second column). Similarly, the relative increase in the efficiency of endocytosis in each mutant was calculated by dividing the endocytosis efficiency in the mutant by the endocytosis efficiency in the wild-type protein as assessed by apical biotinylation as described above (Table I, third column). If the apical content was decreased by the same factor as the endocytosis efficiency was increased, then the enhanced endocytosis would be the only reason for the lowered apical content in this mutant. However, in all mutants, the apical content was decreased to a greater extent than the endocytosis efficiency. For example, in the N5 mutant, the apical content was decreased by 4.5-fold, but the internalization efficiency was increased by only 2-fold. This indicates that the apical membrane delivery was also affected by the mutation because the relative apical content reflects a balance between apical membrane delivery and endocytosis. If the apical content in the mutant is decreased by X-fold compared with the wild-type protein and the endocytosis efficiency is increased by Y-fold, then the apical membrane delivery rate must be decreased by Z = X/Y-fold. Thus, as shown in Table I, the effect of each mutation on the apical sorting efficiency was calculated as a ratio between the factor in the second column, (CA/CT)wt/(CA/CT)mut, and the factor in the third column, (endocytosis efficiency)mut/(endocytosis efficiency)wt.Table IEffect of N-glycosylation site mutations on the relative apical content, endocytosis, and apical sorting of mutant YFP-β compared with wild-type YFP-βYFP-βDecrease in apical content normalized by total complex-type (CA/CT)aCA, YFP-β on the apical membrane; CT, complex-type glycosylated YFP-β in the cell lysate; ND, not detectable.Increase in endocytosis efficiencyDecrease in apical sorting efficiency (calculated)bDecrease in apical sorting efficiency = (decrease in CA/CT)/(increase in endocytosis efficiency).-fold-fold-foldN16.23.12.0N21.21.21.0N35.22.62.0N41.41.11.3N54.52.02.3N61.61.11.4N7NDNDNDWild-type1.01.01.0a CA, YFP-β on the apical membrane; CT, complex-type glycosylated YFP-β in the cell lysate; ND, not detectable.b Decrease in apical sorting efficiency = (decrease in CA/CT)/(increase in endocytosis efficiency). Open table in a new tab The apical sorting efficiency was decreased in all the mutants except N2 up to 2.3-fold (Table I). The very low content of complex-type YFP-β in N7 did not allow determination of the relative apical content in the cell line expressing this mutant. Effect of Inhibitors of ER Glucosidase and Golgi Mannosidases I and II on Sorting and Trafficking of YFP-β in LLC-PK1 Cells—Treatment of cells with castanospermine, an inhibitor of the ER glucosidase, completely blocked oligosaccharide trimming and did not allow glycoprotein processing, as shown in Fig. 1. As a result, YFP-β detected in the presence of castanospermine has a higher molecular mass than t