title: Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway.
abstract: Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.
authors.affiliation: Department Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Lindsey.Costantini@phd.einstein.yu.edu
authors.forename: Lindsey M
authors.initials: LM
authors.lastname: Costantini
authors.forename: Oksana M
authors.initials: OM
authors.lastname: Subach
authors.forename: Matias
authors.initials: M
authors.lastname: Jaureguiberry-bravo
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
authors.forename: Erik L
authors.initials: EL
authors.lastname: Snapp
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D055571:Secretory Pathway
chemical_list: D008164:Luminescent Proteins; C409504:blue fluorescent protein, Aequorea victoria; D003545:Cysteine