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Research Article 2775 A phenylalanine-based folding determinant in intestinal sucrase-isomaltase that functions in the context of a quality control mechanism beyond the endoplasmic reticulum Marcus J. Pröpsting1, Heike Kanapin1, Ralf Jacob1,2 and Hassan Y. Naim1,* 1 Department of Physiological Chemistry, University of Veterinary Medicine Hannover, 30559 Hannover, Germany Department of Cytobiology, Philipps-University of Marburg, 35037 Marburg, Germany 2 *Author for correspondence (e-mail: [email protected]) Accepted 9 March 2005 Journal of Cell Science 118, 2775-2784 Published by The Company of Biologists 2005 doi:10.1242/jcs.02364 Journal of Cell Science Summary Phenotype II of congenital sucrase-isomaltase deficiency in man is characterized by a retention of the brush border protein sucrase-isomaltase (SI) in the ER/cis-Golgi intermediate compartment (ERGIC) and the cis-Golgi. The transport block is due to the substitution of a glutamine by a proline at amino acid residue 1098 that generates a temperature-sensitive mutant enzyme, SIQ1098P, the transport of which is regulated by several cycles of anterograde and retrograde transport between the ER and the cis-Golgi (Propsting, M. J., Jacob, R. and Naim, H. Y. (2003). J. Biol. Chem. 278, 16310-16314). A quality control beyond the ER has been proposed that implicates a retention signal or a folding determinant elicited by the Q1098P mutation. We have used alanine-scanning mutagenesis to screen upstream and downstream regions flanking Q1098 and identified a putative motif, F1093-x-F1095x-x-x-F1099 that is likely to be implicated in sensing the folding and subsequent trafficking of SI from the ER to the Golgi. The characteristics of this motif are three phenylalanine residues that upon substitution by alanine generate the temperature-sensitive SIQ1098P phenotype. This mutant protein undergoes transport arrest in the ERGIC and cis-Golgi compartments and acquires correct folding and functional activity at reduced temperatures as a consequence of cycles of anterograde and retrograde transport between the ER and cis-Golgi. Other amino acid residues in this motif are not significant in the context of phenotype II. We propose that the phenylalanine cluster is required for shielding a folding determinant in the extracellular domain of SI; substitution of a Q by a P at residue 1098 of sucrase disrupts this determinant and elicits retention of SIQ1098P in ERGIC and cis-Golgi in phenotype II of CSID. Introduction A physiological malfunction in many genetic disorders is elicited by an altered folding determinant in a protein due to a mutation in the gene leading, for example, to an intracellular block, missorting or degradation of this protein (Stein et al., 2002; Bross et al., 1999). Normally, membrane and secretory proteins that have reached a native, folded and assembled structure are capable of exiting the endoplasmic reticulum and are then transported to their target organelles and compartments within the cell. The cell has exploited quality control mechanisms that distinguish between correctly folded and unfolded proteins and these mechanisms are therefore essential in modulating intracellular transport or degradation if they do not fold (Brodsky and McCracken, 1999; Ellgaard et al., 1999; Sitia and Braakman, 2003). Therefore, the quality control system provides a stringent and precise discriminating system that provides a structural and functional cellular integrity. Until very recently, it was thought that the quality control machinery is exclusive to the endoplasmic reticulum, with many censoring components, such as molecular chaperones, sugar residues and catalysts of disulphide bond formation (Ellgaard and Helenius, 2001; Ellgaard et al., 1999; Hammond and Helenius, 1994; Vashist et al., 2001). In fact, many artificially generated mutants of membrane and secretory proteins, as well as naturally occurring mutants implicated in genetic diseases, were shown not to traverse the quality control machinery of the ER but to be retained in that organelle (Hebert et al., 1997; David et al., 1996; Jacob et al., 2002; Letourneur et al., 1995; Nehls et al., 2000). The exception to this rule is found in two cases of the intestinal disorder, congenital sucrase-isomaltase deficiency (CSID). CSID is an autosomal recessive intestinal disease that is characterized by the absence of the sucrase and most of the maltase digestive activity within the sucrase-isomaltase (SI) enzyme complex, with the isomaltase activity varying from absent to normal (Treem, 1995). Clinically, the disease is manifested as an osmotic-fermentative diarrhoea upon ingestion of disaccharides and oligosaccharides. Analysis of this disorder at the molecular and subcellular levels has unravelled a number of phenotypes of CSID, which are Key words: Sucrase-isomaltase, ER-quality control, cis-Golgi, ERGIC, Protein folding, Phenylalanine-based motif 2776 Journal of Cell Science 118 (12) Table 1. A list of the mutants generated by alanine-scanning mutagenesis Mutant Plasmid for biochemical/ confocal analysis Amino acid sequence and position Ala1 Ala2 Ala3 Ala4 Ala5 Ala6 Ala21 Ala22 Ala31 Ala32 Ala33 Ala3Q Ala41 QP pSIAla1/pSIAla1-YFP pSIAla2/pSIAla2-YFP pSIAla3/pSIAla3-YFP pSIAla4/pSIAla4-YFP pSIAla5/pSIAla5-YFP pSIAla6/pSIAla6-YFP pSIAla21/pSIAla21-YFP pSIAla22/pSIAla22-YFP pSIAla31/pSIAla31-YFP pSIAla32/pSIAla32-YFP pSIAla33/pSIAla33-YFP pSIAla3Q/pSIAla3Q-YFP pSIAla41/pSIAla41-YFP pSIQP/pSIQP-YFP L1089G1090P1091 → A1089A1090A1091 G1092F1093 → A1092A1093 F1095D1096Q109 → A1095A1096A1097 F1099I1100Q11101 → A1099A1100A1101 I1102S1103T11104 → A1102A1103A1104 R1106L1107P1108 → A1106A1107A1108 G1094A F1095A F1098A N1096A D1097A N1096D1097Q1098 → A1096A1097A1098 F1099A Q1098P Mutagenesis primer 5′-3′ forward* gctatttgggattctgcggcagctggatttgcttttaat gattcttggctgccggcagctgcttttaatgac ctgcctggatttgctgcagctgcccagttcattcaaatatc gcttttaatgaccaggcagctgcaatatcgactcgcc gaccagttcattcaagcagctgctcgcctgccatcag cattcaaatatcgactgcagcggcatcagaatatatatatgg gggattcttggctacctgcatttgcttttaatgacc gggattcttggctacctggagctgcttttaatgacc ctgcctggatttgctgctaatgaccaattcattcaaatatc ctgcctggatttgcttttgctgaccaattcattcaaatatc ctgcctggatttgctttcaatgcccagttcattcaaatatc ctggatttgcttttgctgcccgcgttcttcaaata gcttttaatgaccaggccattcaaatatcgact cctggatttgctttcaatgacccattcattcaaatatcgac Journal of Cell Science *The reverse primers were the complementary sequences of the forward primers. characterized by perturbations in the intracellular transport, polarized sorting, aberrant processing and defective function of SI (Fransen et al., 1991; Jacob et al., 2000c; Ritz et al., 2003; Sterchi et al., 1990; Naim et al., 1988). The enzyme SI is a type II transmembrane glycoprotein from the brush border of intestinal epithelial cells, where it is involved in the hydrolysis of disaccharides (Semenza, 1986). SI is synthesized in the ER, processed in the Golgi apparatus and transported directly to the apical membrane (Matter et al., 1990). To reach this final destination, SI has to pass several control points that ensure transport of only properly folded proteins (Rothman and Orci, 1992). Soon after synthesis in the ER several chaperones cooperate with modification enzymes such as protein disulphide isomerase and glycosyl transferases to generate properly folded molecules (Ellgaard and Helenius, 2001). A quality control mechanism retains improperly folded molecules in the ER until they have acquired a proper folding, or directs them to the proteasome for degradation (Brodsky and McCracken, 1999). However, phenotype II of congenital SI deficiency (CSID) does not conform to this general paradigm. In this case SI exits the ER and is retained in the ER/cis-Golgi intermediate compartment (ERGIC) and the cisGolgi (Fransen et al., 1991; Ouwendijk et al., 1996). The deficiency is based on a point mutation in the SI gene that results in a substitution of a glutamine by proline at amino acid residue 1098 of the sucrase subunit (Q1098P) (Moolenaar et al., 1997; Ouwendijk et al., 1996). This mutation is found in a region that has strong homologies with some members of the family of glycosyl hydrolases (Moolenaar et al., 1997; Naim et al., 1991; Ouwendijk et al., 1998) in mammalian cells and also in the yeast Schwanniomyces occidentalis. Strikingly, substitution of the glutamine residue with proline at the corresponding position of human lysosomal α-glucosidase, resulted in its retention in ERGIC and cis-Golgi (Moolenaar et al., 1997) thus generating a similar phenotype to that of SI (denoted throughout SIQ1098P). Escape from the ER and accumulation of this mutant in the cis-Golgi and ERGIC compartments is suggestive of the existence of a quality control mechanism operating beyond the ER. A most important characteristic of this mutant phenotype is its temperature sensitivity and acquisition of correct folding at a permissive temperature 25°C (Propsting et al., 2003). The underlying mechanism of this folding behaviour involves several cycles of retrograde and anterograde trafficking between the ER and the cis-Golgi until the protein has attained the correct folded structure. Key players in this mechanism are the ER molecular chaperones calnexin and the immunoglobulin binding protein, BiP, and a putative retention signal of the protein in the cis-Golgi (Propsting et al., 2003). Here, mutant SI binds BiP and calnexin and then sequential binding to these chaperones takes place. The protein is then brought to the cis-Golgi where it is blocked and brought back to the ER, presumably through calnexin bound to mutant SI at this stage. Similar steps occur until the protein has acquired correct folding, after which it can no longer be blocked in the cis-Golgi. One possible hypothesis to explain this retention implicates a subdomain flanking the conserved glutamine residue at position 1098 in the transport of SI along the secretory pathway. The exchange of this glutamine, or specific residues within this domain, would alter the folding of SI and expose peptides that are recognized in the cis-Golgi, resulting in the retention of SI in this compartment. This scenario was examined in the work presented here by alanine scanning mutagenesis of regions flanking glutamine 1098. The biosynthetic features, temperature sensitivity and subcellular localization of these mutants were assessed. We examined in detail a region between amino acids 1093 and 1099 in which three phenylalanines play a central role in constituting a potential retention signal for cis-Golgi. Materials and Methods Materials Streptomycin, penicillin, glutamine, Dulbecco’s modified Eagle’s medium (DMEM), methionine-free DMEM (denoted Met-free medium), Fetal calf serum (FCS) and trypsin were purchased from BioWest, Essen, Germany. Pepstatin, leupeptin, aprotinin, trypsininhibitor and molecular mass standards for SDS-PAGE were purchased from Sigma, Deisenhofen/Germany. Soybean trypsin inhibitor was obtained from Roche Diagnostics, Mannheim, Germany. L-[35S]methionine (>1000 Ci/mmol) and protein A-Sepharose were obtained from Amersham Pharmacia Biotech, Freiburg, Germany. Acrylamide, N,N′-methylenebisacrylamide and N,N,N′,N′tetramethylenediamine (TEMED) were purchased from Carl Roth GmbH, Karlsruhe, Germany. Sodium dodecyl sulphate (SDS), ammonium persulphate, dithiothreitol and Triton X-100 (TX-100) were obtained from Merck, Darmstadt/Germany. Restriction enzymes Protein quality control beyond the ER 2777 were obtained from MBI Fermentas, St. Leon-Rot, Germany and Isis-polymerase was obtained from Qbiogene, Heidelberg, Germany. Journal of Cell Science Immunochemical reagents For immunoprecipitation of human SI a mixture of the mouse monoclonal antibodies (mAb) of hybridoma HBB 1/219, HBB 2/619 and HBB 3/705 was used (Hauri et al., 1985). Anti-ER-Golgi intermediate compartment mAb ERGIC53 was a product of hybridoma G1/93 (Schweizer et al., 1988). Antibodies against GM130 and calreticulin were obtained from BD Bioscience, Heidelberg, Germany. The secondary antibody Alexa Fluor 633 was purchased from Invitrogen Laboratories, Inc, Heidelberg, Germany. Calnexin and BiP were precipitated with the polyclonal antibodies pAb-calnexin and pAb-BiP. Fig. 1. Schematic drawing of the sucrase-isomaltase alanine scanning-mutants. The primary sequence of the SI polypeptide between amino acids W1088 and S1108 is depicted in single letter code together with the SI cDNA sequence. Q1098 is indicated in bold; this residue is substituted by P in phenotype II of CSID. The Ala triplets (Ala1 to Ala6) are boxed and shown underneath the corresponding mutated SI sequences. The individual Ala mutants are shown underneath the corresponding substituted amino acid residue. Another Ala triplet was constructed to encompass Q1098 and two upstream residues, N1096 and D1097 (denoted Ala3Q). The Ala triple or single mutants in grey boxes are responsible for the phenotype II of CSID. Construction of cDNA clones Mutation of the plasmids pSI-YFP (Jacob and Naim, 2001) and pSG8-SI (Ouwendijk et al., 1996) were generated by oligonucleotide directed mutagenesis with the Quick Change™ in vitro Mutagenesis System from Stratagene (Table 1). The mutations were confirmed by sequencing. The expression plasmids are indicated by pSI followed by a suffix corresponding to the Ala triplet or the Ala single mutant and YFP (yellow fluorescent protein) in the case of fusion proteins (for example: pSIAla1 or pSIAla1-YFP) (see Table 1). Transient transfection of COS1 cells, biosynthetic labelling and immunoprecipitation COS-1 cells were transiently transfected with various plasmids encoding the SI-cDNA (Table 1) by Fig. 2. Expression of SI, SIQ1098P and SIAla1-6 alanine scanning mutants in COS-1 cells at 37°C and 20°C. COS1 cells were transiently transfected with the respective expression plasmids as indicated. Forty-eight hours after transfection the cells were pulsed with [35S]methionine for 30 minutes at 37°C followed by different chase periods at 37°C or 20°C. After cell lysis, SI was immunoprecipitated with mAb antiSI and the immunoprecipitates were treated with endo H, endo F/GF or left untreated. They were subjected to SDS-PAGE on 5% slab gels and analysed by phosphorimaging. For some gels, longer exposure was used because of the faint band intensity. using DEAE-dextran essentially as described previously (Jacob et al., 2000a). 48 hours after transfection, the cells were biosynthetically labelled. The cells were incubated in methionine-free MEM 2778 Journal of Cell Science 118 (12) Journal of Cell Science containing 50 µCi of [35S]methionine for the indicated time intervals and chased in pulse-chase experiments with non-labelled methionine for different periods of time. Subsequently, the cells were rinsed twice with ice-cold PBS and solubilized with 1 ml lysis buffer containing 25 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.5% deoxycholate (DOC) and 0.5% TX-100 supplemented with 1 mM PMSF, 1 µg/ml pepstatin, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml antipain and 50 µg/ml trypsin inhibitor for 30 minutes at 4°C. After 1 hour of preclearing with 30 µl of protein A-Sepharose, the immunoprecipitation was performed with the mAb antiSI mix and 50 µl of protein A-Sepharose as described previously (Jacob et al., 2000b). In some experiments the cells were labelled at 20°C and chased for prolonged periods of times. SDS-PAGE and deglycosylation analysis The immunoprecipitates were further processed on SDSPAGE according to the method of Laemmli (Laemmli, 1970). The apparent molecular masses were assessed by comparison with high molecular mass markers (Sigma, Deisenhofen, Fig. 3. Colocalization of SI mutants with ER, ERGIC and cis-Golgi in transfected COS-1 cells. COS-1 cells were transfected with pSI Ala1-6-YFP and incubated at 37°C (A-C) for 48 hours before fixation. In some experiments the cells were cultured at 20°C 1 day post-transfection and then overnight at 20°C followed by a temperature shift to 37°C for 4 hours (D). The subcellular localization of the YFP fusion proteins was monitored utilizing antibodies directed against calreticulin (A), ERGIC 53 (B) and GM130 (C). The cells were analysed by confocal microscopy on a Leica TCS-SP2 microscope. YFP fluorescence is indicated in green and a secondary Alexa Fluor 633-conjugated antibody was used for immunodetection of the compartment-specific antibodies (red). Arrows indicate YFP fluorescence on the plasma membrane. n, nucleus. Bars, 25 µm. Germany) run on the same gel. In some experiments, deglycosylation of the immunoprecipitates with endo-N-acetylglucosaminidase H (endo H) and endo-β-N-acetylglucosaminidase F/glycopeptidase F (endo F) (both from Roche Diagnostics, Mannheim, Germany) was performed prior to SDS-PAGE analysis as described previously (Naim et al., 1987). After electrophoresis, the gels were fixed and Protein quality control beyond the ER 2779 Fig. 4. Expression of SIAla21, SIAla22, SIAla31, SIAla32, SIAla33 and SIAla41 in COS-1 cells at 37°C and 20°. COS-1 cells were transiently transfected with pSIAla21, pSIAla22, pSIAla31, pSIAla32, pSIAla33 or pSIAla41, labelled and processed in a pulse-chase experiment with [35S]methionine as described for Fig. 2. The deglycosylated and control samples were subjected to SDS-PAGE prior to scanning by a phosphorimaging device. For some gels longer exposure was used because of the faint band intensity. analysed on a phosphorimaging device (BioRad, Munich, Germany). Journal of Cell Science Confocal laser fluorescence microscopy The cellular localization of expressed proteins in COS-1 cells was studied with cells grown on coverslips. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Saponin. immunolabelling was carried out using mAb anti-ERGIC-53, mAb anti-calreticulin or mAb anti-GM130. The secondary antibody Alexa Fluor 633 conjugated rabbit anti-mouse was used for fluorescence detection. Confocal images of fixed cells were acquired 2 days after transfection using a Leica TCS SP2 microscope with an 63 water planapochromat lens (Leica Microsystems). Dual colour Alexa Fluor 633 and YFP images were obtained by sequential scans with the 633 nm lines of an HeNe laser and 514 nm excitation lines of an argon laser and the optimal emission wavelength for Alexa 633 or YFP, respectively, as previously described (Jacob et al., 2002). For the assessment of temperature-sensitive characteristics the cells were cultured 1 day after transfection at 20°C for almost 18 hours (overnight) and the temperature was then raised to 37°C for 4 hours. Fig. 3C,D. See previous page for legend. Results The Q1098P substitution in phenotype II of CSID generates a temperaturesensitive SIQ1098 mutant enzyme, whose intracellular transport is regulated by several cycles of anterograde and retrograde transport between the ER and the cis-Golgi (Propsting et al., 2003). A quality control beyond the ER has been proposed that prevents misfolded proteins from being further transported along the secretory pathway to the cell surface, and implicates a retention signal elicited by the Q1098P mutation. We therefore set out to analyse short subdomains in the direct neighbourhood of Q1098 by alanine scanning mutagenesis of several blocks upstream and 2780 Journal of Cell Science 118 (12) Journal of Cell Science downstream of this particular amino acid residue. Fig. 1 shows that the region from position L1089 to P1107 was first scanned by six alanine triple blocks (denoted SIAla1 to SIAla6). Two triplets were generated that span the central position 1098 and eight single amino acid exchanges in the A2, A3 and A4 block were introduced for a detailed analysis of the distinct amino acids. Each mutant was transiently transfected into COS-1 cells and analysed 48 hours after transfection in biosynthetic labelling experiments at 37°C. Fig. 2 shows that SIAla1, SIAla5 and SIAla6 revealed similar biosynthetic features to wild-type SI, exemplified by the conversion of the mannose-rich endo H-sensitive 210 kDa pro-SIh polypeptide to the endo H-resistant form of 245 kDa (pro-SIc; SIc, complex glycosylated SI) in pulse-chase experiments. By contrast, the alanine-triple mutants SIAla2, SIAla3 and SIAla4 persisted predominantly as mannose-rich polypeptides throughout the entire chase time and thus were similar the SIQ1098P mutant phenotype (Fig. 2). More importantly, these mutants acquired endo H resistance, i.e. complex glycosylation when the cells were labelled at 20°C in a fashion similar to SIQ1098P, indicating that these mutants possess similar temperaturesensitive characteristics to SIQ1098P (Fig. 2). To further investigate whether these mutants express the same phenotype as SIQ1098P the subcellular localization of YFP variants of these mutants was examined using markers of the ER, ERGIC and cis-Golgi; calreticulin, ERGIC 53 and GM130, respectively. The alanine-triplets SIAla2, SIAla3 and SIAla4 revealed features similar to those of SIQ1098P. Thus, these mutants accumulate intracellularly in perinuclear regions typical of the ER as assessed by colocalization with calreticulin (Fig. 3A) and colocalized also with ERGIC 53 (Fig. 3B) and the cis-Golgi marker GM 130 (Fig. 3C). Finally, they were not found in vesicular structures or at the cell surface. Culturing of the cells expressing SIAla2, SIAla3 and SIAla4 at 20°C followed by a shift to 37°C leads to the appearance of these mutants at the cell surface, reminiscent of a transportcompetent configuration similar to the Fig. 5. Colocalization of SIAla21, SIAla22, SIAla31, SIAla32, SIAla33 and SIAla41 with ER, ERGIC and cis-Golgi in transfected COS-1 cells. The subcellular localization of fluorescent alanine scanning mutants of SI and intracellular markers was monitored as described for Fig. 3. Arrows indicate YFP fluorescence on the plasma membrane. n, nucleus. Bars, 25 µm. Protein quality control beyond the ER Journal of Cell Science SIQ1098P phenotype (Fig. 3D shows the data obtained with the mutant triplet Ala3 as an example representing the other two Ala-triplets, Ala2 and Ala4). Altogether, the biochemical and confocal analyses clearly show that SIQ1098P and the alanine blocks, SIAla2, SIAla3 and SIAla4 share common phenotypical characteristics indicating that the domain of SI that is responsible for a cis-Golgi block in the CSID Q1098P phenotype encompasses a stretch of ten amino acids from G1092 to Q1101 (Fig. 1). To precisely define the amino acid residues involved in generating the phenotype II within these blocks we performed a second series of alanine scans in which each individual amino acid residue in these blocks was substituted by alanine. This series included the mutants SIAla21, SIAla22, SIAla31, SIAla32, Fig. 5C,D. See previous page for legend. 2781 SIAla33 and SIAla41 (Fig. 1). The A1094 residue in the A2 block was not further analysed, since it occurs as alanine in the wildtype protein. Pulse-chase analyses indicate that the mutants SIAla21, SIAla32 and SIAla33 were transport competent, acquired complex glycosylated forms and as such resembled the wildtype protein (Fig. 4). In contrast, three other mutants, SIAla22, SIAla31 and SIAla41 revealed characteristics similar to the SIQ1098P phenotype, since they persisted as mannose-rich glycosylated forms at 37°C (Fig. 4). We examined the temperature-sensitive features of SIAla22, SIAla31 and SIAla41 and performed pulse-chase analyses of these mutants at 20°C. Here again, processing of these mutants to complex glycosylated endo H-resistant species was achieved, pointing to a similar phenotype as that of SIQ1098P (Fig. 4). Confocal microscopy of these mutants corroborated the biochemical data and showed that these mutants were localized in the ER (Fig. 5A), ERGIC (Fig. 5B) and cis-Golgi (Fig. 5C), based on their colocalization with the protein markers calreticulin, ERGIC 53 and GM130. The temperature-sensitivity of these three mutants was also analysed by culturing the transfected cells at 20°C. Here again, and in line with the biochemical data, the mutants were detected at the cell surface after a temperature shift to 37°C (Fig. 5D shows the data obtained with the mutant SIAla31, as an example representing the other two mutants, SIAla22 and SIAla41). In a fashion similar to the SIQ1098P mutant, enzymatic activity of the SIAla31, SIAla22 and SIAla41 mutants could be restored at this permissive temperature. Obviously, the SIAla31, SIAla22 and SIAla41 mutants show similar structural and functional features to the naturally occurring mutant SIQ1098P, in which the acquisition of normal trafficking and function utilizes several cycles of anterograde and retrograde steps between the endoplasmic reticulum and the Golgi, implicating the molecular chaperones calnexin and BiP (Propsting et al., 2003). We therefore investigated the cellular mechanism that is responsible for the retention of mutations in the F1093-xF1095-x-x-x-F1099-motif by studying their interaction with two components of the quality control system in the ER, BiP and calnexin. Firstly, the interaction of these chaperones with SIAla1, a mutant with comparable structural features to wildtype SI, was compared to that of the transport incompetent SIAla31 mutant. Here, transfected COS-1 cells were biosynthetically labelled with [35S]methionine continuously for 6 hours and the detergent extracts were immunoprecipitated with anti-BiP or Journal of Cell Science 2782 Journal of Cell Science 118 (12) Fig. 6. Sequential interaction of SIQ/P with BiP and calnexin. COS-1 cells were transfected with pSG8-Ala1 and pSG8-Ala31. (A) Fortyeight hours post-transfection the cells were biosynthetically labelled at 37°C for 6 hours followed by cell lysis and immunoprecipitation of SI. The cell lysates were immunoprecipitated with anti-BiP or anti-calnexin and subsequently with mAb anti-SI. (B) Transiently transfected COS-1 cells were labelled for 30 minutes followed by different chase intervals at 20°C. The cell lysates were immunoprecipitated sequentially with mAb anti-SI and anti-BiP or anti-calnexin. The samples were analysed with SDS-PAGE and phosphoimaging. anti-calnexin antibodies, followed by subsequent precipitation of the bound material with mAb anti-SI. Fig. 6A demonstrates a weak interaction of SIAla1 with BiP and calnexin while the mannose-rich form of the SIAla31 mutant bound more strongly to both chaperones. The kinetics of this interaction was further analysed in pulse-chase experiments. Fig. 6B shows an early interaction of the SIAla31 mutant with BiP and calnexin within 30 minutes of the pulse. Following 30 minutes of chase the interaction with BiP diminished and the binding to calnexin increased at the same time to a maximum. This binding pattern was reversed after 60 minutes of chase. At this time more SIAla31 molecules were found associated with BiP than with calnexin, indicating that the underlying retention mechanism of SI mutants in the F1093-x-F1095-x-x-x-F1099-motif is based on cycles of association, dissociation and re-association with the ER resident proteins BiP and calnexin. Comparable kinetics of interaction with BiP and calnexin were obtained with the other two mutants SIAla22 and SIAla41 (data are not shown). Strikingly, the mutated residues at positions 1093, 1095 and 1099 that have generated the CSID phenotype II are exclusively phenylalanines. Obviously, these residues play a central role in the transport competence of SI within the sequence F1093-xF1095-x-x-x-F1099, whereby x can be any amino acid; alterations in this motif are not tolerated along the secretory pathway of SI between the ER and the Golgi, since they result in the retention of SI as a mannose-rich glycosylated polypeptide in the ERGIC or cis-Golgi compartments (Figs 4 and 5). What is the role of the Q1098 residue in this context? We have previously shown by site-directed mutagenesis of Q1098 that the intracellular transport of SI to the cell surface is not affected by Q1098 (Ouwendijk et al., 1998). We corroborated these data by alanine scanning at Q1098 itself. Fig. 7A demonstrates that the processing profiles of SI carrying Q1098A were similar to the wild-type SI protein, exemplified by the mannose-rich and complex glycosylated forms appearing at 4 hours of pulse labelling. Likewise, confocal analysis revealed predominant cell surface and Golgi labelling of the Q1098A mutant similar to wild-type SI (Fig. 7B). Other SI mutants carrying exchanges of Q1098 by residues from other amino acid groups, E, Y and N did not reveal a notable alteration in the transport behaviour (data are essentially similar to those shown in Fig. 7A and are therefore not shown). We finally asked whether Q1098 acts in the context of a short motif, thereby implicating sequences in its immediate vicinity. For this an Ala-triplet was generated to substitute the sequence N1096D1097Q1098 (indicated SIAla3a). Here again, a wild-type phenotype was obtained at the biochemical and subcellular levels (Fig. 7B). Altogether, the data unequivocally indicate that the Q1098 is not an essential residue in the F1093-x-F1095-x-x-xF1099 sequence and it is likely that its substitution by a P in the CSID phenotype has drastically altered the secondary structure of this motif. Discussion The retention of a cell surface protein such as intestinal brush border sucrase-isomaltase in the ERGIC and cis-Golgi compartments strongly suggests the existence of putative quality control mechanisms operating beyond the ER. The structural and functional characteristics of SI in phenotype II of CSID, SIQ1098P, provide strong support to this notion. Thus, correct folding, competent intracellular transport, and full enzymatic activity can be restored by expression of this mutant at the permissive temperature of 20-25°C by utilizing several cycles of anterograde and retrograde steps between the endoplasmic reticulum and the Golgi apparatus. In addition to the CSID phenotype II a growing body of information about other secretory and membrane proteins supports the concept of a quality control operating beyond the ER. In combined deficiency of factors V and VIII, an autosomal recessive bleeding disorder, the post-ER protein, ERGIC-53 or LMAN1, has been proposed to regulate the transport from ER to Golgi of these coagulation factors (Cunningham et al., 2003; Nichols et al., 1998). Another example is that of the GPI-anchored tissue-non-specific alkaline phosphatase, in which a single amino acid exchange from N153 to D153 in a naturally occurring mutation causes cis-Golgi retention (Ito et al., 2002). Together with our observations of phenotype II the consensus is now emerging that multiple quality control checkpoints, including those in the ER, do exist along the secretory pathway that regulates the trafficking to the cell surface but that there are recognition signals and interacting protein components. In this work we have identified a consensus signal, F1093-xF1095-x-x-x-F1099, that may function as a sensor for correct trafficking of SI to the cell surface. The destruction of this motif by the Q1098P mutation in CSID is sufficient to elicit retention of SI in the cis-Golgi and the ERGIC compartments. Interestingly, a similar motif is found in two other membrane glycoproteins, the mammalian lysosomal α-glucosidase and glucoamylase of the Schwanniomyces occidentalis yeast (Ouwendijk et al., 1996; Naim et al., 1991). Strikingly, mutagenesis of the glutamine in this motif to a proline in lysosomal α-glucosidase generates a protein that is blocked in the cis-Golgi and ERGIC in a fashion similar to SI, lending strong support to the notion that this motif is functional in the Journal of Cell Science Protein quality control beyond the ER 2783 Fig. 7. Expression profile and subcellular localisation of SIQ1098A and SIAla3Q in COS-1 cells. (A) The cDNAs of the SIQ1098A and the Ala triplet AlaNDQ mutants were transfected into COS-1. The cells were biosynthetically labelled for 4 hours with [35S]methionine, lysed and immunoprecipitated with mAb anti-SI antibodies. The samples were analysed by SDS-PAGE and phosphorimaging. (B) COS-1 cells were transfected with the cDNAs corresponding to the Ala mutants SIQ1098A and SIAla3Q to which YFP was fused (denoted pSIQ1098A-YFP and pSIAla3Q-YFP) and fixed 48 hours after transfection for immunofluorescence staining with calreticulin-, ERGIC 53- and GM130specific antibodies. Alexa Fluor 633-conjugated antibody was used for immunodetection of the compartment-specific antibodies as indicated in Fig. 3. Arrows indicate YFP fluorescence on the plasma membrane. n, nucleus. Bars, 25 µm. context of a different protein and that this motif is likely to be implicated in a quality control beyond the ER. One may postulate that the phenylalanine cluster in the extracellular domain of SI is required for shielding of a putative cis-Golgi or ERGIC retention signal that is hidden in the polypeptide. Disruption of the F1093-x-F1095-x-x-x-F1099 structure by a mutation such as Q1098P in CSID drastically alters the secondary structure of this sequence and exposes this retention signal. This sequence may function, therefore, to sense the correct folding of SI beyond the ER and is implicated in an anterograde and retrograde transport of mutant SI between the ER and the cis-Golgi. Only when the protein has acquired adequate folding after several cycles of binding and dissociation to BiP and calnexin is its further transport to the cell surface warranted. Phenylalanine residues that are implicated in ER export have so far been found on the cytosolic domains of transmembrane proteins (Nufer et al., 2002; Otte and Barlowe, 2002). The membrane protein ERGIC-53 (or LMAN1) carries a C-terminal di-phenylalanine motif that is required for efficient ER export and mediates COPII binding (Nufer et al., 2002). In addition, many G-protein receptors harbour the sequence F(x)6LL in their cytosolic domains for ER export and proper folding of the polypeptide (Duvernay et al., 2004). According to these observations a second role of the phenylalanine cluster in the F1093-x-F1095-x-x-x-F1099 motif could be the proper folding of the enzyme, generating a three-dimensional transport competent that can bypass the quality control mechanism in the ER and reach the cell surface. In one of the most prominent diseases involving protein trafficking, the CFTR channel is misfolded because of the loss of one single phenylalanine (CFTRdelta508) (Cheng et al., 1990). As a consequence, the mutant is unable to transit from the ER to the plasma membrane. This effect can be overcome by compounds known to stabilize proteins in their native conformation (Brown et al., 1997). Further evidence on the stabilizing effect of phenylalanine comes from studies on the headpiece subdomain of villin (Frank et al., 2002). Here a cluster of three conserved phenylalanine residues forms a hydrophobic core that may stabilize aromatic-aromatic interactions (Burley and Petsko, 1985). Any replacement of these residues with leucine results in destabilization of the domain. The three aromatic amino acids in the F1093-x-F1095-x-x-x-F1099 motif could serve a similar purpose in the folding process of SI. In particular, secondary structures such as alpha helices and beta strands are stabilized by aromatic side chain interactions (Meurisse et al., 2004). Furthermore, our observations directly suggest protein retention at the level of the cis-Golgi complex. In a classical view, the ER quality control machinery is located at the exit site of the ER (Ellgaard and Helenius, 2001). Recent data have provided evidence for sequential checkpoint mechanisms of ER quality control along the passage from ER to Golgi (Vashist et al., 2001; Vashist and Ng, 2004). Early checkpoints would then encompass the chaperones BiP and calnexin/calreticulin (Molinari and Helenius, 2000), while ERGIC-53 is involved in the trafficking from ER to Golgi (Nichols et al., 1998). In this case malfolded SI mutants would pass these early checkpoints until they reach the cis-Golgi. Here, a retrieval mechanism would package the enzyme into COPIcoated transport vesicles and recycle them back to the ER. We thank Hans-Peter Hauri (Biozentrum, University of Basel, Switzerland), Erwin Sterchi (University of Bern, Switzerland) and Dallas Swallow (University College London, UK) for the gifts of 2784 Journal of Cell Science 118 (12) antibodies against SI, and Hans-Peter Hauri for the ERGIC-53 antibody. This work was supported by a grant from the Deutsche Forschungsgemeinschaft, DFG (grant no. 331/1-3/1-4 to H.Y.N.). Journal of Cell Science References Brodsky, J. L. and McCracken, A. A. (1999). ER protein quality control and proteasome-mediated protein degradation. Semin. Cell Dev. Biol. 10, 507-513. Bross, P., Corydon, T. J., Andresen, B. S., Jorgensen, M. M., Bolund, L. and Gregersen, N. (1999). Protein misfolding and degradation in genetic diseases. Hum. Mutat. 14, 186-198. Brown, C. R., Hong-Brown, L. Q. and Welch, W. J. (1997). Correcting temperature-sensitive protein folding defects. J. Clin. Invest. 99, 1432-1444. Burley, S. K. and Petsko, G. A. (1985). 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