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. 1991 Jun;173(11):3539–3546. doi: 10.1128/jb.173.11.3539-3546.1991

Removal of N-glycosylation sites of the yeast acid phosphatase severely affects protein folding.

M A Riederer 1, A Hinnen 1
PMCID: PMC207969  PMID: 2045373

Abstract

The influence of N glycosylation on the production of yeast acid phosphatase was studied. A set of synthetic hypoglycosylation mutants was generated by oligonucleotide-directed mutagenesis of the 12 putative sequons (Asn-X-Ser/Thr). Derepression of the hypoglycosylation mutants and analysis of their molecular sizes showed that all 12 sequons of the wild-type acid phosphatase are glycosylated. Activity measurements in combination with pulse-chase experiments revealed that the specific activity was not impaired by the introduced amino acid exchanges. However, absence of N glycosylation severely affected protein folding. Protein folding was found to be the rate-limiting factor in acid phosphatase secretion, and improper folding resulted in irreversible retention of malfolded acid phosphatase in the endoplasmic reticulum. With a decreasing number of attached glycan chains, less active acid phosphatase was secreted. Efficiency of correct folding was shown to be temperature dependent; i.e., lower temperatures could compensate for the reduction in attached oligosaccharides. In addition, protein folding and stability were shown to depend on both the number and the position of the attached oligosaccharides. N glycosylation was found to occur in a process independent of secondary structures, and thus our data support the model of a cotranslocational mechanism of glycosylation.

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