Studying the Protein Quality Control System of <em>D. discoideum</em> Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Max-Planck Institute for Molecular Cell Biology and Genetics

Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.

Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates.

Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition. Here, we examine how disease-causing and normal conformations are linked, and discuss the possibility that the dynamic order of the cytoplasm emerges, at least in part, from the collective properties of intrinsically disordered prion-like domains. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.

Protein disorder, prion propensities, and self-organizing macromolecular collectives.

Many unicellular organisms age: as time passes, they divide more slowly and ultimately die. In budding yeast, asymmetric segregation of cellular damage results in aging mother cells and rejuvenated daughters. We hypothesize that the organisms in which this asymmetry is lacking, or can be modulated, may not undergo aging.

Fission yeast does not age under favorable conditions, but does so after stress.

One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell.

Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation.

Many protein-misfolding diseases are caused by proteins carrying prion-like domains. These proteins show sequence similarity to yeast prion proteins, which can interconvert between an intrinsically disordered and an aggregated prion state. The natural presence of prions in yeast has provided important insight into disease mechanisms and cellular proteostasis. However, little is known about prions in other organisms, and it is not yet clear whether the findings in yeast can be generalized. Using bioinformatics tools, we show that Dictyostelium discoideum has the highest content of prion-like proteins of all organisms investigated to date, suggesting that its proteome has a high overall aggregation propensity. To study mechanisms regulating these proteins, we analyze the behavior of several well-characterized prion-like proteins, such as an expanded version of human huntingtin exon 1 (Q103) and the prion domain of the yeast prion protein Sup35 (NM), in D. discoideum. We find that these proteins remain soluble and are innocuous to D. discoideum, in contrast to other organisms, where they form cytotoxic cytosolic aggregates. However, when exposed to conditions that compromise molecular chaperones, these proteins aggregate and become cytotoxic. We show that the disaggregase Hsp101, a molecular chaperone of the Hsp100 family, dissolves heat-induced aggregates and promotes thermotolerance. Furthermore, prion-like proteins accumulate in the nucleus, where they are targeted by the ubiquitin-proteasome system. Our data suggest that D. discoideum has undergone specific adaptations that increase the proteostatic capacity of this organism and allow for an efficient regulation of its prion-like proteome.

Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation.

RNA-protein (RNP) granules have been proposed to assemble by forming solid RNA/protein aggregates or through phase separation into a liquid RNA/protein phase. Which model describes RNP granules in living cells is still unclear. In this study, we analyze P bodies in budding yeast and find that they have liquid-like properties. Surprisingly, yeast stress granules adopt a different material state, which is reminiscent of solid protein aggregates and controlled by protein disaggregases. By using an assay to ectopically nucleate RNP granules, we further establish that RNP granule formation does not depend on amyloid-like aggregation but rather involves many promiscuous interactions. Finally, we show that stress granules have different properties in mammalian cells, where they show liquid-like behavior. Thus, we propose that the material state of RNP granules is flexible and that the solid state of yeast stress granules is an adaptation to extreme environments, made possible by the presence of a powerful disaggregation machine.

Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules.

Prion-like proteins can undergo conformational rearrangements from an intrinsically disordered to a highly ordered amyloid state. This ability to change conformation is encoded in distinctive domains, termed prion domains (PrDs). Previous work suggests that PrDs change conformation to affect protein function and create phenotypic diversity. More recent work shows that PrDs can also undergo many weak interactions when disordered, allowing them to organize the intracellular space into dynamic compartments. However, mutations within PrDs and altered aggregation properties have also been linked to age-related diseases in humans. Thus, the physiological role of prion-like proteins, the mechanisms regulating their conformational promiscuity and the links to disease are still unclear. Here, we summarize recent work with prion-like proteins in Dictyostelium discoideum. This work was motivated by the finding that D. discoideum has the highest content of prion-like proteins of all organisms investigated to date. Surprisingly, we find that endogenous and exogenous prion-like proteins remain soluble in D. discoideum and do not misfold and aggregate. We provide evidence that this is due to specific adaptations in the protein quality control machinery, which may allow D. discoideum to tolerate its highly aggregation-prone proteome. We predict that D. discoideum will be an important model to study the function of prion-like proteins and their mechanistic links to disease.

Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem.

Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be associated with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromolecular assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mechanical stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiological states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state.

Liliana Malinovska

Max-Planck Institute for Molecular Cell Biology and Genetics

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging