The images ( e) correspond to the time point at 4 h after the beginning of the chase, while the quantifications in ( f) correspond to the lifetime calculation, fitting entire chase curves as in Figure 3 (see below). ( e) Block of proteasome function with lactacystin (LCY) and respective quantifications ( f), showing that inhibiting proteasome function increases the lifetime of SNAP targeted to the nucleus or the ER, but does not affect its mitochondrial turnover. ( d) Image intensity quantification in 75 images revealing a drop in SNAP-TMR-BG intensity after the use of CHX compared to untreated cells for all three conditions. ( c) Representative images of cells expressing NLS, mitochondrial inner membrane, and actin (Lifeact) constructs, blocked with SNAP-Cell Block for 1 h, followed by a 3-h pulse in the absence (untreated control) or the presence of cycloheximide (CHX, blocking protein synthesis). The black arrowhead shows the selected concentration for further experiments (0.2 µM, representative image shown in ( a). ( b) Similar approach as in ( a) with different concentrations of SNAP-Cell Block revealing a significant drop in TMR-BG intensity. ( a) Representative images of cells expressing the mitochondrial inner membrane construct, either blocked with SNAP-Cell Block for 1 h or left untreated, followed by a 10 min pulse with SNAP-Cell TMR-BG. Testing the reliability of the assay for the optical analysis of protein turnover. Rab5a SNAP-tag optical analysis of protein turnover protein stability pulse-chase. Overall, our data reveal that both changes in protein localization and functional state are key modulators of protein turnover, and protein lifetime fluctuations can be considered to infer changes in cellular behavior. Finally, we followed up on the increased lifetime observed for the constitutively active Rab5a (Q79L), and we found that its stabilization correlates with enlarged endosomes and increased interaction with membranes. We also tested a selection of mutants modulating the function of three extensively studied proteins, the Ca 2+ sensor calmodulin, the small GTPase Rab5a and the brain creatine kinase (CKB). With this approach, we obtained precise measurements of protein turnover rates in several subcellular compartments. For this purpose, we used an imaging approach based on the pulse-labeling of 17 representative SNAP-tag constructs for measuring protein lifetimes. Here, we have considered two factors influencing protein turnover: the subcellular localization of a protein and its functional state. While the regulators of proteostasis are the machineries controlling protein production, folding and degradation, several other factors can influence this process. This equilibrium avoids the accumulation of potentially toxic proteins, which could lead to cellular stress and death. Protein homeostasis is an equilibrium of paramount importance that maintains cellular performance by preserving an efficient proteome.
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