title: Potential pitfalls and solutions for use of fluorescent fusion proteins to study the lysosome.
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.
authors.affiliation: . Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, United States of America ; Department of Pharmacology Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey, United States of America.
authors.forename: Ling
authors.initials: L
authors.lastname: Huang
authors.affiliation: . Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, United States of America ; Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey, United States of America.
authors.forename: Douglas
authors.initials: D
authors.lastname: Pike
authors.affiliation: . Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, United States of America ; Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey, United States of America.
authors.forename: David E
authors.initials: DE
authors.lastname: Sleat
authors.affiliation: . Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, United States of America ; Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey, United States of America.
authors.forename: Vikas
authors.initials: V
authors.lastname: Nanda
authors.affiliation: . Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, United States of America ; Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey, United States of America.
authors.forename: Peter
authors.initials: P
authors.lastname: Lobel
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000009:adverse effects; D006031:Glycosylation / Q000187:drug effects; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure*; D051379:Mice; D018613:Microscopy, Confocal; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000009:adverse effects; D057057:Serine Proteases / Q000737:chemistry / Q000378:metabolism; D046509:Tissue Culture Techniques; D000091346:Tripeptidyl-Peptidase 1; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D000626:Aminopeptidases; D017931:DNA Primers; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D057057:Serine Proteases; D000091346:Tripeptidyl-Peptidase 1; D033921:Vesicular Transport Proteins; D000097573:Red Fluorescent Protein; C497045:Npc2 protein, mouse; C000717268:Tpp1 protein, mouse