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.
Highlights
Lysosomes are membrane-delimited, acidic organelles present in essentially all eukaryotic cells [1]
In the course of studies investigating the molecular basis for lysosomal cholesterol efflux, we sought to develop a fluorescent Niemann-Pick disease type C protein 2 (NPC2) fusion protein that retained function and integrity in the lysosome. mCherry was chosen primarily due to its resistance to acid quenching [13]
An initial fusion protein was constructed with the C-terminal Ser of mouse NPC2 linked via the peptide RARDPPVAT, which is encoded by the plasmid multiple cloning site, to the N-terminal Met of mCherry (Fig. 1A, NPC2-9aa-mCherry)
Summary
Lysosomes are membrane-delimited, acidic organelles present in essentially all eukaryotic cells [1] This compartment contains over 70 hydrolytic enzymes [2,3] that function to degrade biological macromolecules taken up by endocytosis, phagocytosis and autophagy. Previous studies have noted that, for some fusion proteins targeted to the lysosome, the reporter tag may be cleaved from the parent protein [8,9]. In some cases, this problem can be addressed by reducing lysosomal protease activity through use of inhibitors or mutant cells deficient in lysosomal proteases [8], though this is not ideal as it may introduce artifacts. Fluorescent tags used for the study of localization must not perturb protein targeting
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