ArticlePloS one2014
Potential pitfalls and solutions for use of fluorescent fusion proteins to study the lysosome.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 43 papers.
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Who cites it
43 citing papers in PubMed, 67 citations in OpenAlex.
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- Directed evolution of genetically encoded LYTACs for cell-mediated delivery.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Two-pore channels regulate endomembrane tension to enable remodeling and resolution of phagolysosomes.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Directed Evolution of Genetically Encoded LYTACs for Cell-Mediated Delivery.bioRxiv : the preprint server for biology · 2023Article
- Mind your tag in single-molecule measurements.Cell reports methods · 2023Article
- Experimental considerations for study of C. elegans lysosomal proteins.G3 (Bethesda, Md.) · 2023Article
- Transcription-Driven Translocation of Cohesive and Non-Cohesive Cohesin In Vivo.Molecular and cellular biology · 2023Article
- BORC-ARL8-HOPS ensemble is required for lysosomal cholesterol egress through NPC2.Molecular biology of the cell · 2022Article
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- Targeting transgenic proteins to alpha granules for platelet-directed gene therapy.Molecular therapy. Nucleic acids · 2022Article
- Dual-fluorescent reporter for live-cell imaging of the ER during DENV infection.Frontiers in cellular and infection microbiology · 2022Article
- RNA-based cooperative protein labeling that permits direct monitoring of the intracellular concentration change of an endogenous protein.Nucleic acids research · 2021Article
- CREG1 promotes lysosomal biogenesis and function.Autophagy · 2021Article
- Identification of conserved slow codons that are important for protein expression and function.RNA biology · 2021Article
- Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection.eLife · 2021Article
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
Abstract
Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6-phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications.
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