Abstract: High-level expression of the GUP1 gene in Saccharomyces cerevisiae resulted in the formation of proliferated structures, which hosted endoplasmic reticulum (ER), Golgi and itinerant proteins. The GUP1 over-expression enhanced ER biogenesis, as shown by the coordinated increased transcription rate of genes involved in both ER and Golgi metabolism and in phospholipids biosynthesis. The formation of Gup1-induced proliferation revealed that it depended on an intact unfolded protein response, because their assembly was reported to be lethal to yeast strains unable to initiate the UPR (Unfolded Protein Response) pathway. GUP1 over-expression affected global ER and Golgi structure and resulted in the biogenesis of novel membrane arrays with Golgi and ER hybrid composition. In fact, a number of ER and Golgi resident proteins together with itinerant proteins that normally cycle between ER and Golgi, were localized in the proliferated stacked membranes. The described assembling of novel membrane structures was affected by the functionality of the Gup1 O-acyltransferase domain, which regulates the Gup1 protein role as remodelase in the glycosylphosphatidylinositol (GPI) anchored proteins biosynthesis. To our knowledge, we presented the first evidence of sub cellular modifications in response over-expression of a GPI-anchor remodelase in S. cerevisiae.CI - Copyright (c) 2010. Published by Elsevier B.V.