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Validating the Golgi two‐hybrid assay's utility in studying glycosylated proteins
Author(s) -
Shew Matthew A.,
Dube Danielle H.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.696.1
Subject(s) - golgi apparatus , glycosylation , secretory pathway , protein–protein interaction , microbiology and biotechnology , two hybrid screening , computational biology , chemistry , biology , biochemistry , yeast , endoplasmic reticulum
Despite its utility in detecting interactions among nuclear and cytosolic proteins, the traditional yeast two‐hybrid assay (Y2H) systematically misses the interactions between secreted and cell surface proteins that traffic through the secretory pathway en route to the outside of the cell. To overcome this limitation, the Kohler lab developed the Golgi two‐hybrid assay (G2H) which tests secretome protein‐protein interactions in the Golgi, where proteins have their full complement of post‐translational modifications. In the G2H, protein‐protein interactions are linked to a selectable parameter within the Golgi ‐ Och1 mannosyltransferase activity. Previous results by Dube and Kohler demonstrate that the G2H can successfully detect the known protein‐protein interaction between MyoD and Id2. Here we set out to validate the G2H's ability to detect the known glycosylation‐dependent interaction between polypeptide N ‐acetylgalactosaminyltransferase‐2 (ppGalNAcT‐2) and its substrate Muc5AC. This work was supported by INBRE and the Camille and Henry Dreyfus Foundation.