Comparative Analyses of Arabidopsis complex glycan1 Mutants and Genetic Interaction with staurosporin and temperature sensitive3a

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.