Investigating the Relationship between N-Linked Glycosylation and Membrane Protein Localization in Saccharomyces cerevisiae

Abstract

Membrane proteins are proteins that are closely associated with the plasma membrane and play a significant role in selective molecular transport, cell-cell recognition, enzymatic activity, and signal transduction. Approximately 25% of all human proteome belongs to membrane proteins, among which G-protein coupled receptors (GPCRs) represent the most prominent family. GPCRs are targets for approximately 60% of marketed drugs for human diseases such as Parkinson's, Alzheimer's, Schizophrenia, cancer, inflammatory diseases, and coronary diseases. However, out of 800 diverse GPCRs in humans, only 24 high-resolution crystal structures have been reported to date, making them one of the most challenging targets in structural biology as they pose significant challenges due to low-level expression, partial hydrophobicity, high conformational flexibility, and instability outside their native lipid bilayer environment. We investigated the relationship between N-linked glycosylation in differential expression and plasma membrane localization patterns when class A GPCRs are expressed in Saccharomyces cerevisiae using experimental methods coupled with bioinformatics for predicting putative N-linked glycosylation sites at the N-terminus of GPCRs. This tandem approach aids in downstream studies that target elucidation of their crystal structures. Brewer's yeast or Baker's yeast, S. cerevisiae, is a suitable expression host for heterologous protein expression as it poses an advanced secretory pathway consisting of cellular machinery that can readily accommodate post-translational modifications (PTMs) like N-linked glycosylation. Herein, a chimera prepared with the N-terminal region of human dopamine D2 (hD2) and human adenosine A2A (hA2 ) was introduced to yeast using the yeast expression plasmid pYES2. The chimera was C-terminally labeled with a green fluorescent protein, ye-GFP, to facilitate monitoring of intracellular trafficking. After introducing this chimera to yeast, yeast cells were induced to express the chimera so that its expression pattern and subcellular localization could be observed using fluorescence microscopy. Moreover, we analyzed the variations in N-termini length of class A GPCRs and putative N-linked glycosylation sites in order to highlight the relationship between N-linked glycosylation in the trafficking and sub-cellular localization of GPCRs.