Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway

Lindsey M. Costantini, Oksana M. Subach, Matias Jaureguiberry-bravo, Vladislav Verkhusha, Erik L. Snapp

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1114-1119
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume430
Issue number3
DOIs
StatePublished - Jan 18 2013

Fingerprint

Secretory Pathway
Glycosylation
Endoplasmic Reticulum
Proteins
Cysteine
Asparagine
Consensus Sequence
Disulfides
Cytoplasm
Technology

Keywords

  • BFP
  • Disulfide bonds
  • Endoplasmic reticulum
  • Fluorescent proteins
  • N-glycosylation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway. / Costantini, Lindsey M.; Subach, Oksana M.; Jaureguiberry-bravo, Matias; Verkhusha, Vladislav; Snapp, Erik L.

In: Biochemical and Biophysical Research Communications, Vol. 430, No. 3, 18.01.2013, p. 1114-1119.

Research output: Contribution to journalArticle

Costantini, Lindsey M. ; Subach, Oksana M. ; Jaureguiberry-bravo, Matias ; Verkhusha, Vladislav ; Snapp, Erik L. / Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway. In: Biochemical and Biophysical Research Communications. 2013 ; Vol. 430, No. 3. pp. 1114-1119.
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