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First published online June 8, 2005
doi: 10.1242/10.1242/jcs.02364

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A phenylalanine-based folding determinant in intestinal sucrase-isomaltase that functions in the context of a quality control mechanism beyond the endoplasmic reticulum

¹ Department of Physiological Chemistry, University of Veterinary Medicine Hannover, 30559 Hannover, Germany
² Department of Cytobiology, Philipps-University of Marburg, 35037 Marburg, Germany

View larger version (15K): [in a new window]   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.

View larger version (84K): [in a new window]   Fig. 2. Expression of SI, SIQ1098P and SIAla1-6 alanine scanning mutants in COS-1 cells at 37°C and 20°C. COS-1 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 anti-SI 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.

View larger version (188K): [in a new window]   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.

View larger version (66K): [in a new window]   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.

View larger version (157K): [in a new window]   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.

View larger version (49K): [in a new window]   Fig. 6. Sequential interaction of SIQ/P with BiP and calnexin. COS-1 cells were transfected with pSG8-Ala1 and pSG8-Ala31. (A) Forty-eight 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.

View larger version (76K): [in a new window]   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 GM130-specific 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.

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