Analysis of ER Resident Proteins in Saccharomyces cerevisiae: Implementation of H/KDEL Retrieval Sequences

Authors

  • Carissa L. Young,

    1. Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA
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  • David L. Raden,

    1. Current address: Department of Biological Sciences, University of Delaware, 330 Wolf Hall, Newark, DE 19716, USA
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  • Anne S. Robinson

    Corresponding author
    1. Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA, USA
    • Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA
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Corresponding author: A. S. Robinson, asr@tulane.edu

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.

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