Almost 4 years ago, I wrote a post on tandem fluorescent timers (tFTs). The idea is to have two different fluorescent proteins fused together to the protein of interest. In the paper from 4 years ago, it was superfolder GFP (sfGFP) and mCherry. sfGFP matures very fast (within minutes) and mCherry matures more slowly (t1/2 ~40min). The ratio beween green to red fluorescent signal indicates the percentage of new vs old proteins, thus acts as a “timer”. This latests paper on tFTs from the same group of Michael Knop’s lab, found that analyzing tFTs might be more complicated due to some possible problems of this system.
Here, they want to test other green FPs: myeGFP (monomeric, yeast optimized enhanced GFP), GFP mut3a, GFPm, Clover and mNeonGreen. Using these green-FP-mCherry timers fused to the same protein (the yeast proton pump Pma1), they could see differnces in red/green ratio that reflected differences in green-FP maturation times, as expected.
However, when they wanted to study degradation kinetics of the different tFTs, they found odd results that were inconsitstent with theoretical calculations. They found that some of the tFTs are only partly degraded, leaving a green-FP or full tFT protein fragment that can still fluoresce and skew the results.
I will not go into all the details of the tests they did, you can read about that in their paper. Suffice to say that they found that green-FPs are poorly degraded. Hence, the position of the tFT in the fusion protein constract, the order by which the green/red FPs are placed (i.e. gree/red or red/green) and the specific folding properties of the green-FP, affect the proteasomal degradation product.
Here are 5 recomendations they suggest for tFT users:
- The time range of the tFT is determined mostly by the slow-maturing FP, but also by the fast maturing and possible FRET between them. Therefore, choose the slow-maturing FP that match the kinetics of the process you study. For the other color – choose the fastest & brightests available.
- If possible, test your tFT in both combinations ( green/red, red/green) for both protein age & protein degradation rates.
- If you use GFP (or any degradation resistant FP), this FP should be distal to the degradation initiation site.
- Although it is prefered to have a tFT that is efficiently degraded, under some circumstances a degradation resistant FP might be better (e.g. to measure fast degrading protein).
- tFTs with inclompete degradation can also be used for systematic analysis of protein half-life, since the ratio of mCherry/sfGFP, for instance, should be a function of the half-life. However, one need to remember to distinguish different degradation pathways.
This is all good advice for any tagging with fluorescent proteins, but particularly when studying protein age/degradation. More so, it highlights the fact that sometimes when we image proteins fused to GFP, what we might be imaging is actually just a fragment fused to GFP.
Khmelinskii A, Meurer M, Ho CT, Besenbeck B, Füller J, Lemberg MK, Bukau B, Mogk A, & Knop M (2016). Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers. Molecular biology of the cell, 27 (2), 360-70 PMID: 26609072