Document Detail


Analysis of ER resident proteins in Saccharomyces cerevisiae: implementation of H/KDEL retrieval sequences.
MedLine Citation:
PMID:  23324027     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Carissa L Young; David L Raden; Anne S Robinson
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2013-02-04
Journal Detail:
Title:  Traffic (Copenhagen, Denmark)     Volume:  14     ISSN:  1600-0854     ISO Abbreviation:  Traffic     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-03-07     Completed Date:  2013-08-15     Revised Date:  2014-04-02    
Medline Journal Info:
Nlm Unique ID:  100939340     Medline TA:  Traffic     Country:  England    
Other Details:
Languages:  eng     Pagination:  365-81     Citation Subset:  IM    
Copyright Information:
© 2013 John Wiley & Sons A/S.
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MeSH Terms
Descriptor/Qualifier:
Endoplasmic Reticulum / metabolism*
Fungal Proteins / genetics,  metabolism
Green Fluorescent Proteins / genetics,  metabolism
HSP40 Heat-Shock Proteins / genetics,  metabolism
HSP70 Heat-Shock Proteins / genetics,  metabolism
Microscopy, Fluorescence
Protein Disulfide-Isomerases / genetics,  metabolism
Protein Sorting Signals*
Protein Transport
Recombinant Fusion Proteins / genetics,  metabolism
Saccharomyces cerevisiae / genetics,  metabolism*
Saccharomyces cerevisiae Proteins / genetics,  metabolism*
Grant Support
ID/Acronym/Agency:
1S10 RR027273-01/RR/NCRR NIH HHS; 5P30RR031160-03/RR/NCRR NIH HHS; 8 P30 GM103519-03/GM/NIGMS NIH HHS; P20 RR015588/RR/NCRR NIH HHS; P20 RR15588/RR/NCRR NIH HHS; P30 GM103519/GM/NIGMS NIH HHS; P30 RR031160/RR/NCRR NIH HHS; R01 GM065507/GM/NIGMS NIH HHS; R01 GM65507/GM/NIGMS NIH HHS; S10 RR027273/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
0/Fungal Proteins; 0/HSP40 Heat-Shock Proteins; 0/HSP70 Heat-Shock Proteins; 0/KAR2 protein, yeast; 0/PDI1 protein, S cerevisiae; 0/Protein Sorting Signals; 0/Recombinant Fusion Proteins; 0/SCJ1 protein, S cerevisiae; 0/Saccharomyces cerevisiae Proteins; 147336-22-9/Green Fluorescent Proteins; EC 5.3.4.1/Protein Disulfide-Isomerases
Comments/Corrections

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