Matthias Peter

Proliferating cells maintain their cytoplasmic density within a narrow range but deviate when entering quiescence or experiencing stress, suggesting active regulation. The mechanisms driving these density adjustments and their impact on cellular function remain unclear. Here, we demonstrate that the conserved cAMP-activated protein kinase A (PKA) is a key regulator of cytoplasmic properties. Inactivation of PKA leads to a drastic increase in cytoplasmic dry mass density and reduced diffusion that depends on the environmental stress response (ESR) transcription factors Msn2/4. This change is mediated by the accumulation of glycogen and trehalose, which have opposing effects on intracellular diffusion. Importantly, the accumulation of these carbohydrates confers stress resistance in distinct ways and independently of their roles as energy sources. Our findings highlight the importance of the biophysical properties of the cytoplasm in stress resistance and the role of glycogen and trehalose in regulating these properties.

Unlike irreversible pathological amyloids, reversible fibrils can be regulated via pH-sensing core motifs, characterized by amyloid properties unleashed upon stress-induced protonation of critical residues. Here, we combined bioinformatic predictions and an in vitro validation pipeline to search for pH-responsive, reversible amyloid core peptides in yeast and human proteomes. This approach uncovered biophysical properties distinguishing pH-sensing and constitutive amyloid cores and established reliable criteria to identify novel reversible assemblies based on sequence data. Selected full-length candidate proteins with evolutionarily conserved pH-sensing motifs were analyzed in Saccharomyces cerevisiae using fluorescence microscopy and SDS-resistance assays, revealing multiple proteins forming reversible assemblies in stationary phase. We found that protonation of a specific histidine in the amyloid core motif of the asparagine synthase Asn1 is necessary and sufficient for assembling catalytically inactive, reversible structures called cytoophidia. Interestingly, mutant cells that fail to assemble Asn1-cytoophidia show defects to recover from stationary phase, demonstrating functional relevance of pH-sensing amyloid core motifs in vivo . Taken together, we uncovered a widespread and conserved pH-sensing mechanism that regulates the reversible assembly and function of structurally diverse fibrils upon stress.

Cellular quiescence is a reversible state essential for survival under nutrient-limiting or growth-restrictive conditions, yet the mechanisms fine-tuning its depth and reversibility remain elusive. Here, we identify Saccharomyces cerevisiae Rts3 as a regulator of the quiescence trajectory downstream of TORC1. Using phosphatase inhibitor beads and mass spectrometry, we characterize Rts3 as a phosphatase interactor in rapamycin-treated cells and define it as an inhibitor of the PP6 phosphatase Sit4. Mechanistically, it employs an α-helix to dock directly into the Sit4-Sap185/190 catalytic cleft. Transcriptionally induced by Gln3/Gat1 during nitrogen starvation, Rts3 is rapidly degraded upon nutrient repletion via a TORC1-SCF ^Cdc4 -proteasome axis. By selectively constraining Sit4-Sap185/190 activity, this inhibitor modulates nitrogen-responsive transcriptional and translational programs to prevent excessive accumulation of Gln3/Rtg3 targets, establishing a feedback loop gating quiescence depth to support long-term survival. Our findings position Rts3 as a dynamic molecular brake on Sit4, ensuring a protective yet reversible quiescent state.

Growth-induced compressive forces regulate diverse biological processes ranging from development to tumor progression, but the underlying molecular mechanisms remain poorly understood. Here, we found that compressive mechanical cues activate the MAP kinase extracellular signal-regulated kinase 5 (ERK5) at the cell periphery, inducing invasive traits in epithelial cells. Using a phospho-proteomic approach combined with an RNAi-based validation screen, we identified several previously unrecognized ERK5 effectors important for cell-cell adhesion and actin organization, among them the myosin light chain (MLC) phosphatase subunit Myosin Phosphatase-Targeting Subunit 1 (MYPT1). Our results demonstrate that ERK5 regulates MYPT1, which in turn inhibits myosin at the cell periphery. ERK5 signaling counteracts Rho-associated protein kinase (ROCK), thereby reducing peripheral actomyosin tension. This relaxation destabilizes cell-cell contacts and promotes cell migration in response to mechanical compression. Together, these findings identify an ERK5-mediated response mechanism activated by compressive force that triggers cell invasion, highlighting ERK5 therapeutic potential.

Cells have evolved multiple strategies to survive environmental stress conditions. This includes the formation of membrane-less cytoplasmic ribonucleoprotein structures called stress granules (SGs) that sequester and protect mRNAs encoding many housekeeping genes. SGs are not static biomolecular condensates but transform into solid states during a maturation phase. Although SG maturation is a hallmark of many neurodegenerative pathologies, little is known about the mechanisms and physiological relevance underlying the maturation process. Here we show that yeast SGs mature into a solid-like state during long-term stationary phase stress, which delays SG disassembly and cell cycle restart. Profiling of phosphorylation sites during stationary phase revealed that SG maturation is driven by protein kinase A (PKA)-dependent phosphorylation of the SG proteome. Indeed, upon stationary phase the catalytic PKA subunits condense in SGs where they maintain kinase activity, while during this time cytoplasmic PKA is inhibited. PKA-mediated phosphorylation of key SG components, like the pyruvate kinase Cdc19, is necessary and sufficient for its timely accumulation in SGs, where Cdc19 assembles into amyloid-like structures. Importantly, inhibiting PKA during long-term stationary phase prevents SG maturation, delaying ordered re-start of cell growth after re-feeding. Taken together, these results describe a SG maturation mechanism, selectively activated during chronic stress, that preserves SG integrity and promotes cell survival. Graphical Highlights: Stress granules mature during chronic stress such as long-term stationary phase Phosphorylation of SG components by PKA is a hallmark of SG maturation Phosphorylation of Cdc19 by PKA promotes its maturation into an amyloid-like state SG maturation acts as a timer for recovery after chronic stress

The human CTLH/GID (hGID) complex emerged as an important E3 ligase regulating multiple cellular processes, including cell cycle progression and metabolism. However, the range of biological functions controlled by hGID remains unexplored. Here, we used proximity-dependent biotinylation (BioID2) to identify proteins interacting with the hGID complex, among them, substrate candidates that bind GID4 in a pocket-dependent manner. Biochemical and cellular assays revealed that the hGID^GID4E3 ligase binds and ubiquitinates ARHGAP11A, thereby targeting this RhoGAP for proteasomal degradation. Indeed, GID4 depletion or impeding the GID4 substrate binding pocket with the PFI-7 inhibitor stabilizes ARHGAP11A protein amounts, although it carries no functional N-terminal degron. Interestingly, GID4 inactivation impairs cell motility and directed cell movement by increasing ARHGAP11A levels at the cell periphery, where it inactivates RhoA. Together, we identified a wide range of hGID^GID4E3 ligase substrates and uncovered a unique function of the hGID^GID4E3 ligase regulating cell migration by targeting ARHGAP11A.

Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-β motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-β-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of β-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a “memory” of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-β structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils.

Ribosome abundance in changing environments is governed by biogenesis and degradation, but the underlying mechanisms regulating these opposing processes remain unknown. Here we show that Suppressor of Tom1 (Stm1), a dormancy factor protecting cytosolic ribosomes during starvation, has an unexpected function to promote ribosome biogenesis during exponential growth conditions. Indeed, Stm1 transiently localizes to the nucleolus and engages with pre-ribosomal particles. Stm1 upregulates transcription of ribosomal protein genes by directly binding the activation domain (AD) of the transcription factor Ifh1. These novel Stm1 functions confer rapamycin-sensitivity and are mediated by its C-terminal intrinsically disordered region (IDR), which is dispensable for ribosome hibernation. We conclude that Stm1 regulates ribosome homeostasis linking ribosome biogenesis and ribosome dormancy.