cDNA library screening to identify interacting proteins of Golgi-localized type II membrane proteins

Abstract

AbstractIn eukaryotes, biosynthesis of many extracellular matrix glycans occurs in the Golgi apparatus. These proteins include glycosyltransferases, modifying enzymes and nucleotide-sugar conversion enzymes, many of which possess a type II membrane topology. Growing evidence indicates that both the function and Golgi localization of many of these proteins are regulated through protein-protein interactions (PPIs). Given the essential nature and conservation of extracellular matrix polysaccharides, it is likely that PPIs are more prevalent among biosynthetic enzymes. However the identification of PPIs among Golgi proteins has been technically challenging due to the generally low abundance and unique membrane topology of these proteins. The aim of this article is to explore the feasibility of cDNA library screening by a yeast-based modified split ubiquitin system as a mean for unbiased screening for PPIs involving a Golgi-localized type II membrane. As a test case, a galacturonosyltransferase1 (GAUT1), involved in pectin biosynthesis in the higher plant Arabidopsis thaliana, was used as the bait. Construction and screening of Arabidopsis cDNA libraries using GAUT1 as the bait successfully led to identification of GAUT7, a previously reported interaction partner of GAUT1, which validates the method. Furthermore, 25 novel candidate interaction partners were identified. The results contribute to shape a field guide for identifying PPIs involving glycan biosynthetic enzymes in eukaryotic cells.