Background

Pulse-chase labelling approaches have been used to study protein turnover for decades. In the long-lived protein degradation (LLPD) assay described here, the proteome of cells in culture is radiolabelled with ¹⁴C valine and chased in order to follow the decline in radioactive proteins as readout of protein degradation. After an initial chase period, the cells are washed to eliminate the degradation products of short-lived proteins, which predominantly result from proteasomal activity. Thereafter, a second chase is initiated, and proper controls are included in order to monitor the autophagic degradation of long-lived proteins. We recently used this method to discover that the calcium-modulating compounds thapsigargin and A23187, which based on results with autophagic markers were previously widely believed to activate autophagy, actually completely block bulk autophagic flux (Engedal et al., 2013). The starting material used in that and previous protein degradation protocols was derived from cells grown in 6-well plates (Bauvy et al., 2009; Engedal et al., 2013) or more (Ronning et al., 1979; Seglen et al., 1979), or involved relatively high amounts of radioactivity (Mizushima et al., 2001; Fuertes et al., 2003a), which is expensive. Recently, we have downscaled and simplified the LLPD protocol to the validated time- and cost-efficient version that we present here.

An overview of the method is shown in Figure 1. To label proteins with radioactive valine, cells are seeded in 24-well plates in complete medium supplemented with ¹⁴C valine. As proteins are synthesised, they incorporate amino acids, including the ¹⁴C valine present in the medium, and therefore the amount of long-lived ¹⁴C valine-labelled proteins increases with time (Figure 1, first part of the curve). Valine is an optimal amino acid to use in the LLPD method, since it is a poorly metabolised amino acid, is well tolerated at high doses, and does not influence autophagy or protein degradation rates (Seglen et al., 1979). Moreover, free valine is readily exchanged over the plasma membrane (Seglen and Solheim, 1978), enabling efficient wash-out of released ¹⁴C valine. After 2-3 days of labelling, unincorporated ¹⁴C valine is removed by a simple wash procedure, and the cells receive new medium supplemented with a high concentration of non-radioactive (‘cold’) valine (‘chase medium’). The large surplus of cold valine prevents reincorporation of released ¹⁴C valine. Thus, from this point on the presence of free ¹⁴C valine is a result of endogenous protein degradation. After a chase period of 18 h, the free ¹⁴C valine that has been produced by degradation of short-lived proteins (predominantly due to proteasomal degradation) is washed out. Next, a second chase period, which we call the ‘sampling period’, is initiated along with experimental treatments and proper controls. Generally, we use a sampling period of 2-6 h to monitor the degradation of long-lived proteins. At the end of the sampling period, trichloroacetic acid (TCA) is added to precipitate intact proteins. The TCA-soluble fraction of degraded proteins (containing free amino acids and small peptides) is separated from the TCA-insoluble fraction (containing intact proteins) by centrifugation, and the radioactivity in each fraction is measured by liquid scintillation counting. This allows calculation of the rate of long-lived protein degradation in the sampling period, expressed as the percentage of radioactivity in the TCA-soluble fraction versus the total amount of radioactivity in the TCA-soluble and–insoluble fractions, divided by the duration of the sampling period (Figure 1).


Figure 1. Overview of the long-lived protein degradation (LLPD) assay. During the labelling period (2-3 days), the amount of radioactive long-lived proteins increases with time. Thereafter, an 18 h chase period allows for degradation of the short-lived proteins and subsequent elimination of the released ¹⁴C valine by a washing step. Consequently, only the degradation of long-lived proteins is followed in the 2-6 h sampling period. Compared to cells kept in complete, nutrient-rich medium (red line), incubating cells with EBSS starvation medium or the mTOR-inhibitor Torin1 produces a very strong degradation of long-lived proteins in the sampling period, due to enhanced bulk autophagy (green line). Autophagic-lysosomal LLPD can be revealed by treatment with the lysosomal inhibitor Bafilomycin A1 (Baf) or RNAi-mediated silencing of key ATGs (siATGs) (yellow line), whereas the contribution to LLPD from the proteasome can be assessed by treatment with proteasomal inhibitors like MG132 (purple line). Blocking both autophagic-lysosomal and proteasomal activity simultaneously will abrogate both main sources of LLPD, thus resulting in minimal loss of ¹⁴C-labelled intact protein (black line). Note that the rise and fall in the curves are schematic and purely intended for illustrative purposes–they are not intended to indicate exact details in the kinetics of long-lived protein labelling and/or degradation. See text for a more detailed description of each of the protocol steps and for representative examples of data.