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Suppose I want to study the trafficking of a peptide throughout the ER, Golgi, and plasma membrane. An idea I had was labeling a secreted or plasma membrane integral protein with GFP and using time-series live cell microscopy to track it through the secretory pathway.

However, I am concerned that by the time everything is set up, the cell will be at steady state, which means GFP signal would be present throughout the secretory pathway, preventing me from doing this tracking.

How might I overcome this, without changing the fundamental technology (GFP reporting)?

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Historically speaking, the use of VSVG (Vesicular Stomatitis Virus G protein) labeled with GFP was indeed a practical method to define the secretory pathway. (@tyersome comment)

However, I am concerned that by the time everything is set up, the cell will be at steady state, which means GFP signal would be present throughout the secretory pathway, preventing me from doing this tracking.

Sec mutations in S.cerevisiae: Many biochemical pathways are initially identified owing to a particular mutation in the synthesis process. Recall Beadle and Tatum experiment and the logic they used to clarify the existence of 3 enzymes used in arginine metabolism. (Image taken From https://www.chegg.com/homework-help/questions-and-answers/following-experiments-beadle-tatum-searching-mutations-disrupt-arginine-biosynthesis-pathw-q41797559)

A similar approach was taken back then to identify the distinct parts of the secretory pathway. Five different mutations in S.cerevisiae were identified named from A to E. Culturing all A to E mutated strains plus VSVG-GFP assay would clearly demonstrate the entire route. (Image taken from https://slideplayer.com/slide/4382105/)

For example, even if the mutated class B, reaches the steady/equilibrated state, transport vesicles, Golgi, and the rest will not show the fluorescence activity (characteristic of VSVG-GFP) because all of the proteins have accumulated in the previous step(i.e budding from Rough ER)

Further Readings :

https://pubmed.ncbi.nlm.nih.gov/2188733/

https://www.sciencedirect.com/science/article/abs/pii/0092867481900647

MCB Lodish et al 8th edition chapter 14 section 14.1

https://www.nature.com/articles/35073068

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  • $\begingroup$ Is this to say that it is indeed impossible to monitor localization without knocking out parts of the pathway and comparing? $\endgroup$ – actinidia Apr 13 at 20:49
  • $\begingroup$ @actinidia Knocking out and comparing is usually the easiest way but, now that we know the basic fundamentals and characteristic features of each step in this pathway, other methods can also be employed but I haven't searched or seen any researches, to be honest. You can search for it on your own. Theoretically, we can use 1) Förster resonance energy transfer (FRET) Biosensors: As you know, there is a pH gradient from ER to cis Golgi and later to trans Golgi. (Later sections are more acidic) So one can use this feature to build a molecular environmental biosensor that assumes different --- $\endgroup$ – Sam Apr 14 at 11:09
  • $\begingroup$ @actinidia ---conformations in the presence of different pHs, which in this case would colorize the pathway with 2 different colors(for example Yellow/Green if GFP and YFP are used in the construction of the biosensor protein.) 2)Pulse-Chase methods definitely could be used but it doesn’t use the fluorescence as you’ve asked, so it is off the table. $\endgroup$ – Sam Apr 14 at 11:10

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