TY - JOUR
T1 - Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway
AU - Costantini, Lindsey M.
AU - Subach, Oksana M.
AU - Jaureguiberry-bravo, Matias
AU - Verkhusha, Vladislav V.
AU - Snapp, Erik L.
N1 - Funding Information:
We thank the Einstein Analytical Imaging Facility for use of the Zeiss Duoscan. This work was supported by grants from the National Institute of General Medical Sciences (NIGMS) (R01GM086530-01) (E.L.S.), National Institute of Diabetes and Digestive and Kidney Diseases NIDDK (5PO1DK041918) (E.L.S), and NIH Training Program in Cellular and Molecular Biology and Genetics Grant T32 GM007491 (L.M.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or the NIH. E.L.S. and L.M.C conceived and designed the study. V.V.V provided expertise and valuable advice. L.M.C, M.J. and O.M.S. performed the experiments. L.M.C and E.L.S analyzed the data and wrote the manuscript. The authors declare no competing financial interests.
PY - 2013/1/18
Y1 - 2013/1/18
N2 - 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.
AB - 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.
KW - BFP
KW - Disulfide bonds
KW - Endoplasmic reticulum
KW - Fluorescent proteins
KW - N-glycosylation
UR - http://www.scopus.com/inward/record.url?scp=84872493952&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84872493952&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2012.12.028
DO - 10.1016/j.bbrc.2012.12.028
M3 - Article
C2 - 23257162
AN - SCOPUS:84872493952
SN - 0006-291X
VL - 430
SP - 1114
EP - 1119
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -