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First published online 15 March 2005
doi: 10.1242/jcs.01740

Journal of Cell Science 118, 1485-1492 (2005)
Published by The Company of Biologists 2005
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Importance of carbohydrate positioning in the recognition of mutated CPY for ER-associated degradation

Zlatka Kostova* and Dieter H. Wolf{ddagger}

Institut für Biochemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany



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  		Fig. 1. Schematic representation of the hypoglycosylated CPY* variants used in this study. (A) The four glycosylation sites of CPY* are numbered according to their positions in the mature enzyme. G255R is the site of the CPY* mutation. (B) Hypoglycosylated CPY* variants are assigned a four-digit binary code, with 1 specifying the presence and 0 the absence of a glycan at a particular position (Asn13, Asn87, Asn168 or Asn368, respectively).

 


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  		Fig. 2. Efficient degradation of CPY* depends on the position of N-glycans. (A) Unglycosylated CPY* is stabilized in wild-type cells. CPY* and CPY*0000 were expressed in a {Delta}prc1 strain under identical conditions. Following pulse-chase, radiolabeled proteins were immunoprecipitated using anti-CPY antiserum. The graph represents the mean of three independent experiments. (B) Pulse-chase analysis of CPY* variants carrying only one N-glycan expressed in a {Delta}prc1 strain. Graphs represent the mean of three independent experiments. A representative autoradiogram is shown for each mutant.

 


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  		Fig. 3. Position rather than number of N-glycans determines the rate of CPY* degradation. Pulse-chase analysis of hypoglycosylated CPY* variants expressed in {Delta}prc1 containing three (A) or two (B) carbohydrate chains shows that only variants lacking the Asn368 glycan (CPY*1100 and CPY*1110) are affected. Graphs represent average data from three independent experiments. (C) A representative autoradiogram is shown for each mutant.

 


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  		Fig. 4. Htm1p functions via the N-glycans of CPY* but it does not exhibit positional preference. (A) Pulse-chase analysis of CPY* and unglycosylated CPY*0000 expressed in isogenic wild-type (HTM1) and {Delta}htm1{Delta}prc1 ({Delta}htm1) strains. (B) Degradation of four hypoglycosylated CPY* species (CPY*0001, CPY*0011, CPY*1100 and CPY*1110) expressed in wild-type (HTM1) and {Delta}htm1{Delta}prc1 ({Delta}htm1) strains was analysed by pulse labeling and immunoprecipitation using anti-CPY antibodies. A representative autoradiogram is shown for CPY* and CPY*0000.

 


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  		Fig. 5. CTG* degradation depends on N-glycans and Htm1p. CTG* and CTG*0000 were expressed from a low-copy-number vector in isogenic wild-type and {Delta}htm1 strains. Proteins were labeled with [35S]-methionine, chased at the indicated time points and immunoprecipitated using anti-CPY antibodies. Data are derived from four (CTG*, {Delta}htm1CTG*0000) or five (CTG*0000, {Delta}htm1CTG*) independent experiments.

 


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  		Fig. 6. The effects of Cne1p on CPY* degradation differ depending on the glycan present. (A,B) Pulse-chase analysis of CPY*, CPY*0000 (A) and CPY*0100 (B) expressed in isogenic wild-type (CNE1) and {Delta}cne1{Delta}prc1 ({Delta}cne1) strains. (C) CPY*0001 degradation was analysed by pulse labeling in isogenic wild-type (CNE1, HTM1), {Delta}cne1{Delta}prc1 ({Delta}cne1) and {Delta}htm1{Delta}prc1 ({Delta}htm1) strains. Data are derived from four independent experiments.

 

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© The Company of Biologists Ltd 2005