title: Comparative analyses of Arabidopsis complex glycan1 mutants and genetic interaction with staurosporin and temperature sensitive3a.
abstract: 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.
authors.affiliation: Institut für Botanik, Westfälische Wilhelms-Universität Münster, 48149 Munster, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Frank
authors.forename: Heidi
authors.initials: H
authors.lastname: Kaulfürst-Soboll
authors.forename: Stephan
authors.initials: S
authors.lastname: Rips
authors.forename: Hisashi
authors.initials: H
authors.lastname: Koiwa
authors.forename: Antje
authors.initials: A
authors.lastname: von Schaewen