Measuring in vivo protein turnover and exchange in yeast macromolecular assemblies

Abstract

Summary We present a comprehensive and robust protocol to track the dynamics of all proteins in a complex in yeast cells. A single member of the protein assembly is tagged and conditionally expressed, minimizing the perturbations to the protein complex. Then, SILAC labeling and affinity purification are used for the assessment of the whole protein complex dynamics. This method can determine and distinguish both subunit turnover and exchange specifically in an assembly to provide a comprehensive picture of assembly dynamics. For complete details on the use and execution of this protocol, please refer to Hakhverdyan et al. (2021) .

Nuclear basket proteins regulate the distribution and mobility of nuclear pore complexes in budding yeast

“…We term this approach d ilution o f RITE -labelled complexes (doRITE). As a proof of concept, we performed doRITE on Nups previously reported to exhibit stable (Nup133, Nup188) or dynamic (Nup60) association with the NPC (Hakhverdyan et al, 2021; Onischenko et al, 2020; Rabut et al, 2004). We imaged the cells at different time points after the induction of recombination (Figure 1E).…”

“…Therefore, we performed doRITE with Mlp1 and Mlp2 to visualize their association dynamics with the NPC in living cells. For comparison, we also performed doRITE with a set of additional Nups from different NPC subcomplexes, which exhibited different dynamic behaviours by metabolic labelling (Hakhverdyan et al, 2021) (Figure 2A).…”

“…Based on FRAP experiments, Mlp1 and Mlp2 were considered to exchange at the NPC within seconds (Niepel et al, 2013). However, a recent metabolic labelling-based biochemical analysis that systematically determined the exchange rate of all Nups on NPCs found the residence half-lives of Mlp1 and Mlp2 to be 4-5 hours (Hakhverdyan et al, 2021). To understand the function and regulation of the nuclear basket and to correctly interpret experimental results on (mis)localization phenotypes, it is crucial to know whether the basket filaments form a stable or dynamic assembly.…”

“…Therefore, we performed doRITE with Mlp1 and Mlp2 to visualize their association dynamics with the NPC in living cells. For comparison, we also performed doRITE with a set of additional Nups from different NPC subcomplexes, which exhibited different dynamic behaviours by metabolic labelling (Hakhverdyan et al, 2021) (Figure 2A). Mlp1, Mlp2 and Pml39 displayed similar behaviour to the stable Nups of group 1 (Nup133, Nup188, Pom152, Nup82, Nup159), which have an exchange half-life of greater than 7 hours at the NPC.…”

An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae

“…Although our lysate intermixing assays indeed show high exchange rates of Brl1-bound NUPs ( Appendix 1—figure 1C ), dynamic exchange within the bait-bound fraction ( Appendix 1—figure 1E , green arrows), which does not have an influence on the labeling in KARMA assays, contributes to the labeling in the intermixing assay. In the future, for more accurate evaluation of labeling kinetics it might be advantageous to minimize post-lysis intermixing by a mild fixation step similar to what has been done before ( Hakhverdyan et al, 2021 ; Subbotin and Chait, 2014 ).

Appendix 1—figure 1.

…”