Final answer:
For studying protein translocation with RFP, it's critical to choose between fusing RFP to the C-terminus or N-terminus based on the protein's final destination, topology, and involvement in the secretory pathway. N-terminal fusion is ideal for proteins whose N-terminus is outside the cell, while C-terminal fusion suits proteins without membrane insertion signals or with mitochondrial targeting at the N-terminus. TagRFP is preferred for its stable fluorescence in low pH conditions.
Step-by-step explanation:
To determine whether to fuse the reporter protein RFP (Red Fluorescent Protein) to the C-terminus or N-terminus of a protein for studying protein translocation, it is important to consider the final destination of the protein and the path it takes through the cell. For proteins targeted to the plant cell wall, which is an acidic compartment, the fusion to the N-terminus or C-terminus can impact the reporter's visibility and function, depending on the protein topology and transmembrane domains. As the sub-cellular localization of protein labeled with a fluorescent protein (FP) poses challenges in a low pH environment, TagRFP is chosen due to its low pKa and stable fluorescence at low pH. If the protein of interest is a transmembrane protein with its N-terminus outside the cell, fusion to the N-terminus could be more appropriate. If the protein does not have a signal for membrane insertion or has a post-translational mitochondrial targeting signal at the N-terminus, then C-terminal fusion might be the better choice.
Furthermore, it must be taken into consideration whether the protein undergoes post-translational modifications, such as core glycosylation, which could potentially interfere with the function or orientation of the RFP reporter if the fusion is on the N-terminus. The protein's passage through the endomembrane system and whether the protein is a permanent resident of the secretory pathway or targeted to the extracellular space also influences the decision on the fusion site.