title: Analysis of ER resident proteins in Saccharomyces cerevisiae: implementation of H/KDEL retrieval sequences.
abstract: An elaborate quality control system regulates endoplasmic reticulum (ER) homeostasis by ensuring the fidelity of protein synthesis and maturation. In budding yeast, genomic analyses and high-throughput proteomic studies have identified ER resident proteins that restore homeostasis following local perturbations. Yet, how these folding factors modulate stress has been largely unexplored. In this study, we designed a series of polymerase chain reaction (PCR)-based modules including codon-optimized epitopes and fluorescent protein (FP) variants complete with C-terminal H/KDEL retrieval motifs. These conserved sequences are inherent to most soluble ER resident proteins. To monitor multiple proteins simultaneously, H/KDEL cassettes are available with six different selection markers, providing optimal flexibility for live-cell imaging and multicolor labeling in vivo. A single pair of PCR primers can be used for the amplification of these 26 modules, enabling numerous combinations of tags and selection markers. The versatility of pCY H/KDEL cassettes was demonstrated by labeling BiP/Kar2p, Pdi1p and Scj1p with all novel tags, thus providing a direct comparison among FP variants. Furthermore, to advance in vitro studies of yeast ER proteins, Strep-tag II was engineered with a C-terminal retrieval sequence. Here, an efficient purification strategy was established for BiP under physiological conditions.
authors: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
authors: Carissa L
authors: CL
authors: Young
authors: David L
authors: DL
authors: Raden
authors: Anne S
authors: AS
authors: Robinson
mesh_terms: D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; C080576:PDI1 protein, S cerevisiae; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C067634:SCJ1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D019704:Protein Disulfide-Isomerases