A sticky situation – pyruvate kinase aggregates when stressed

Stress. It affects all of us in a variety of different ways. Some of us more so than others. The important factor though is how to deal with it. For me, I wind down with my favourite tunes and a large bar of galaxy, but for an individual cell, they have a much more sophisticated approach tailored to different sorts of stress. A common response is the formation of stress granules; membraneless organelles composed of an aggregation of proteins and RNA, serving to prevent further protein production when conditions in the cell are not favourable. Recently, Saad et al. (1) found in yeast that pyruvate kinase, a key protein in energy production, reversibly aggregates in stressful conditions into the granules and the regulation of the process.

The stress of a cell

Whilst we may stress over finding a new job, an upcoming exam and just life in general, we also share some of the stresses of single cells like yeast. These include extreme temperatures, starvation due to lack of glucose (our energy source) and lack of nitrogen, needed for protein production.

Pyruvate kinase is an enzyme important in the breakdown of glucose to produce ATP (the source of power in a cell*). It is responsible for the conversion of phosphoenolpyruvate (PEP) to pyruvate, one of many stages in this process. The enzyme’s activity is enhanced when bound to a substrate upstream in this pathway, fructose 1-6 bisphosphate (F16BP). However, when glucose levels are low, F16BP levels also plummet effectively ‘shutting off’ pyruvate kinase.

When Saad’s group observed yeast during this stressful condition, pyruvate kinase concentrated into foci, believed to be stress granules (Figure 1). The same occurred when the yeast were entering the stationary growth phase and on heat-shock but not during nitrogen starvation. This strongly suggests pyruvate kinase’s association with stress granules is a stress-specific stress adaptation.

*To learn more about ATP see https://wordpress.com/read/blogs/98184563/posts/181

cell stress.png
Figure 1: Pyruvate kinase distribution in normal vs. ‘stressed’ conditions e.g when low glucose levels


Low complexity regions

The importance of membraneless organelles, like stress granules, in cells are becoming ever more apparent as well as their formation. In particular it is thought a key feature of the formation involves proteins containing low complexity regions (LCRs). Pyruvate kinase contains a LCR between its catalytic domain and F16BP binding site.

Four sites in its LCR are phosphorylatable. When mutagenesis prevented phosphorylation there was an increased stress granule formation. Conversely, when the four sites were mutated to aspartate, acting as a phosphomimic, pyruvate kinase remained soluble and unaggregated. Since the LCR is buried when pyruvate kinase is in its tetrameric form, it is thought that phosphorylation prevents aggregation during exposure of LCRs when the tetrameric complex temporally dissociates.

Phosphorylation and metabolite regulation

Glucose breakdown is big business for a cell. Therefore, everything needs to be tightly regulated. This may be why in addition to dephosphorylation triggering aggregation, it was found that F16BP plays a role. When pyruvate kinase was mutated stopping F16BP from binding, the aggregates persisted even when glucose levels were restored, basically suggesting that F16BP binding is essential to revert to the ‘normal’ state (Figure 1). Phosphorylation and metabolite binding thus work together to coordinate the dynamic changes between the mobile and solid-aggregate state.

Since LCRs are seen in 44% proteins, reversible aggregation under different cellular conditions may represent a widespread phenomenon. This stresses that protein aggregation is not all bad news but raises further questions of how cells deal with distinguishing pathophysiological aggregates caused by misfolding from physiological ones helping the yeast in its time of need.

For more on membraneless organelle formation read this blog post (https://sciencelife.uchospitals.edu/2017/03/09/molecules-form-gels-to-help-cells-sense-and-respond-to-stress/) or check out how LCRs are involved in transcription with my earlier blog post (https://asheekeyscienceblog.com/2017/09/09/get-transcribed-in-style-the-ctd-that-everyones-talking-about/)

Even Further Reading

(1)  Saad et al. Reversible protein aggregation is a protective mechanism to ensure cell cycle restart after stress. Nature Cell Bio, 19, 1202 -1213 (2017)

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