Pulsatile dynamics in the yeast proteome

Abstract

The activation of transcription factors in response to environmental conditions is fundamental to cellular regulation. Recent work has revealed that some transcription factors are activated in stochastic pulses of nuclear localization, rather than at a constant level, even in a constant environment [1-12]. In such cases, signals control the mean activity of the transcription factor by modulating the frequency, duration, or amplitude of these pulses. Although specific pulsatile transcription factors have been identified in diverse cell types, it has remained unclear how prevalent pulsing is within the cell, how variable pulsing behaviors are between genes, and whether pulsing is specific to transcriptional regulators or is employed more broadly. To address these issues, we performed a proteome-wide movie-based screen to systematically identify localization-based pulsing behaviors in Saccharomyces cerevisiae. The screen examined all genes in a previously developed fluorescent protein fusion library of 4,159 strains [13] in multiple media conditions. This approach revealed stochastic pulsing in ten proteins, all transcription factors. In each case, pulse dynamics were heterogeneous and unsynchronized among cells in clonal populations. Pulsing is the only dynamic localization behavior that we observed, and it tends to occur in pairs of paralogous and redundant proteins. Taken together, these results suggest that pulsatile dynamics play a pervasive role in yeast and may be similarly prevalent in other eukaryotic species.

Figure 1. A 4-phase screen identifies 9 pulsing proteins, all of which are transcriptional regulators

(A) Pulsing is defined as the coherent translocation of many molecules of a protein in and out of an organelle in response to a constant input. (B) Time-lapse movies of the GFP library were acquired at low time resolution (∼51 minutes) under four environmental conditions. (C) Examination of these low time-resolution movies revealed 170 potential pulsing proteins. (D) Repeats of movies of these 170 proteins at higher time resolution showed 64 pulsing proteins. (E) 25 of these 64 pulsing proteins were cell-cycle related while (F) 31 were due to focal drift, leaving 9 pulsing proteins remaining. 7 pulsing proteins are sequence-specific transcriptional regulators while 2 are histone deacetylase complex members. Scale bars are 2 μm. Times are indicated in minutes.

Figure 2. A transcription factor screen confirms 10 pulsing transcription factors

(A-L) Filmstrips of 12 transcription factors reveal pulses in nuclear localization across varying timescales and conditions. Many, but not all of the proteins that pulse have a duplicate or redundant protein that also pulses. These proteins are grouped accordingly in grey boxes. Filmstrips are labeled with the protein name and condition. Scale bars are 2 μm. Times are indicated in minutes.

Figure 3. Pulsing is variable

(A-L) Single-cell traces show that pulses vary from cell to cell (across colors on the same trace), from paralog to paralog (across columns) and from protein to protein. All traces are from the same movie that generated corresponding filmstrips in Figure 2. Scale bars are 2 μm. Times are indicated in minutes. All traces have been smoothed.