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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Comparative Analyses of Arabidopsis complex glycan1 Mutants and Genetic Interaction with staurosporin and temperature sensitive3a^1,[W],[OA]

Institut für Botanik, Westfälische Wilhelms-Universität Münster, 48149 Munster, Germany (J.F., H.K.-S., S.R., A.v.S.); and Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, and Molecular and Environmental Plant Science Program, Texas A&M University, College Station, Texas 77843–2133 (H.K.)

We compare three Arabidopsis (Arabidopsis thaliana) complex glycan1 (cgl1) alleles and report on genetic interaction with staurosporin and temperature sensitive3a (stt3a). STT3a encodes a subunit of oligosaccharyltransferase that affects efficiency of N-glycan transfer to nascent secretory proteins in the endoplasmic reticulum; cgl1 mutants lack N-acetyl-glucosaminyltransferase I activity and are unable to form complex N-glycans in the Golgi apparatus. By studying CGL1-green fluorescent protein fusions in transient assays, we show that the extra N-glycosylation site created by a point mutation in cgl1 C5 is used in planta and interferes with folding of full-length membrane-anchored polypeptides in the endoplasmic reticulum. Tunicamycin treatment or expression in the stt3a-2 mutant relieved the folding block, and migration to Golgi stacks resumed. Complementation tests with C5-green fluorescent protein and other N-glycosylation variants of CGL1 demonstrated that suppression of aberrant N-glycosylation restores activity. Interestingly, CGL1 seems to be functional also as nonglycosylated enzyme. Two other cgl1 alleles showed splicing defects of their transcripts. In cgl1 C6, a point mutation affects the 3' splice site of intron 14, resulting in frame shifts; in cgl1-T, intron 11 fails to splice due to insertion of a T-DNA copy. Introgression of stt3a-2 did not restore complex glycan formation in cgl1 C6 or cgl1-T but suppressed the N-acetyl-glucosaminyltransferase I defect in cgl1 C5. Root growth assays revealed synergistic effects in double mutants cgl1 C6 stt3a-2 and cgl1-T stt3a-2 only. Besides demonstrating the conditional nature of cgl1 C5 in planta, our observations with loss-of-function alleles cgl1 C6 and cgl1-T in the stt3a-2 underglycosylation background prove that correct N-glycosylation is important for normal root growth and morphology in Arabidopsis.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Antje von Schaewen (schaewen{at}uni-muenster.de).

^[W] The online version of this article contains Web-only data.

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www.plantphysiol.org/cgi/doi/10.1104/pp.108.127027

^* Corresponding author; e-mail schaewen{at}uni-muenster.de.

Received July 25, 2008; accepted August 24, 2008; published September 3, 2008.