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. 2017 Aug 18;7:8788. doi: 10.1038/s41598-017-09173-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Introducing N-glycosylation sites in yeast Kar2 severely impairs its function. The wild-type yeast strain KAR2 and the temperature sensitive mutant kar2-159 were transformed with an empty vector (p), or vectors for the expression of Kar2, Kar2-HA, variant Kar2-HA introducing sequons 1 through 7 (see text for details), or Kar2-HA sequon-revertants 1.Q, 3.Q, or 5.Q, as indicated. In sequon revertants, the introduced sequons encoding Asn were replaced by the codon encoding Gln. (A) Liquid cultures of transformed KAR2 and kar2-159 strains or (B) kar2-159 strains transformed with KAR2-HA glycosylation variants were grown overnight, serially diluted, spotted onto SC-URA plates and grown at the indicated temperatures and times. (C) anti-HA western blot from kar2-159 transformed with wild type KAR2-HA or glycosylation variants 1–7. (D) and (E) Protein extracts with or without EndoH treatment, separated through SDS-PAGE, and blotted with anti-HA antibody. (ung) is unglycosylated, (g) is glycosylated, (unt) is untranslocated. (F) Cartoon structures of Kar2 showing the location of introduced glycosylation sequons that were glycosylated. N-terminal and C-terminal domains are depicted in blue and pink ribbons, respectively.