SEP 16, 2020 12:45 PM EDT

GFP Complementation Screening of the Yeast Inner Nuclear Membrane Proteome



The inner nuclear membrane of eukaryotic cells defines many aspects of gene expression and chromatin organization.  It is also therapeutically important as several diseases, termed “laminopathies” are related to the function of the inner nuclear membrane.  Nevertheless, the intimate connection with the outer nuclear membrane and the endoplasmic reticulum make it difficult to biochemically probe the proteome of the inner nuclear membrane without contamination.  We have leveraged the power of yeast genomics and split-GFP complementation to highlight only the inner nuclear membrane facing epitopes and performed a candidate screen of ~1000 genes in living yeast cells.  To our surprise, ~400 genes showed significant localization at the inner nuclear membrane.  A counter-screen with degradation pathway mutants revealed that the INMAD pathway and Asi1 is the dominant influence on the INM proteome.  In addition, we have performed low throughput follow up studies of protein-protein interactions unique to this compartment via fluorescence cross-correlation spectroscopy.  These have revealed unique molecular interactions in this compartment that can provide insight into unique roles of traditional ER proteins at the inner nuclear membrane.