Peroxisome biogenesis initiated by protein phase separation.

Peroxisomes are organelles that carry out ß-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction1. Among disease-causing variant genes are those required for protein transport into peroxisomes. The peroxisomal protein import machinery, which also shares similarities with chloroplasts2, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes3. Current models postulate a large pore formed by transmembrane proteins4; however, so far, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes the membrane proteins Pex13 and Pex14 and the cargo-protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid-liquid phase separation (LLPS) with Pex5-cargo. Intrinsically disordered regions in Pex13 and Pex5 resemble those found in nuclear pore complex proteins. Peroxisomal protein import depends on both the number and pattern of aromatic residues in these intrinsically disordered regions, consistent with their roles as 'stickers' in associative polymer models of LLPS5,6. Finally, imaging fluorescence cross-correlation spectroscopy shows that cargo import correlates with transient focusing of GFP-Pex13 and GFP-Pex14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time frames, suggesting that they may form channels at different saturating concentrations of Pex5-cargo. Our findings lead us to suggest a model in which LLPS of Pex5-cargo with Pex13 and Pex14 results in transient protein transport channels7.

Reg1 and Snf1 regulate stress-induced relocalization of protein phosphatase-1 to cytoplasmic granules.

The compartmentalization of cellular function is achieved largely through the existence of membrane-bound organelles. However, recent work suggests a novel mechanism of compartmentalization mediated by membraneless structures that have liquid droplet-like properties and arise through phase separation. Cytoplasmic stress granules (SGs) are the best characterized and are induced by various stressors including arsenite, heat shock, and glucose deprivation. Current models suggest that SGs play an important role in protein homeostasis by mediating reversible translation attenuation. Protein phosphatase-1 (PP1) is a central cellular regulator responsible for most serine/threonine dephosphorylation. Here, we show that upon arsenite stress, PP1's catalytic subunit Glc7 relocalizes to punctate cytoplasmic granules. This altered localization requires PP1's recently described maturation pathway mediated by the multifunctional ATPase Cdc48 and PP1's regulatory subunit Ypi1. Glc7 relocalization is mediated by its regulatory subunit Reg1 and its target Snf1, the AMP-dependent protein kinase. Surprisingly, Glc7 granules are highly specific to arsenite and appear distinct from canonical SGs. Arsenite induces potent translational inhibition, and translational recovery is strongly dependent on Glc7, but independent of Glc7's well-established role in regulating eIF2α. These results suggest a novel form of stress-induced cytoplasmic granule and a new mode of translational control by Glc7.

SDS22 selectively recognizes and traps metal-deficient inactive PP1.

The metalloenzyme protein phosphatase 1 (PP1), which is responsible for ≥50% of all dephosphorylation reactions, is regulated by scores of regulatory proteins, including the highly conserved SDS22 protein. SDS22 has numerous diverse functions, surprisingly acting as both a PP1 inhibitor and as an activator. Here, we integrate cellular, biophysical, and crystallographic studies to address this conundrum. We discovered that SDS22 selectively binds a unique conformation of PP1 that contains a single metal (M2) at its active site, i.e., SDS22 traps metal-deficient inactive PP1. Furthermore, we showed that SDS22 dissociation is accompanied by a second metal (M1) being loaded into PP1, as free metal cannot dissociate the complex and M1-deficient mutants remain constitutively trapped by SDS22. Together, our findings reveal that M1 metal loading and loss are essential for PP1 regulation in cells, which has broad implications for PP1 maturation, activity, and holoenzyme subunit exchange.

New ubiquitin-dependent mechanisms regulating the Aurora B-protein phosphatase 1 balance in Saccharomyces cerevisiae.

Protein ubiquitylation regulates many cellular processes, including cell division. We report here a novel mutation altering the Saccharomyces cerevisiae E1 ubiquitin-activating enzyme (uba1-W928R) that suppresses the temperature sensitivity and chromosome loss phenotype of a well-characterized Aurora B mutant (ip1-2). The uba1-W928R mutation increases histone H3-S10 phosphorylation in the ipl1-2 strain, indicating that uba1-W928R acts by increasing Ipl1 activity and/or reducing the opposing protein phosphatase 1 (PP1; Glc7 in S. cerevisiae) phosphatase activity. Consistent with this hypothesis, Ipl1 protein levels and stability are elevated in the uba1-W928R mutant, likely mediated via the E2 enzymes Ubc4 and Cdc34. In contrast, the uba1-W928R mutation does not affect Glc7 stability, but exhibits synthetic lethality with several glc7 mutations. Moreover, uba1-W928R cells have an altered subcellular distribution of Glc7 and form nuclear Glc7 foci. These effects are likely mediated via the E2 enzymes Rad6 and Cdc34. Our new UBA1 allele reveals new roles for ubiquitylation in regulating the Ipl1-Glc7 balance in budding yeast. While ubiquitylation likely regulates Ipl1 protein stability via the canonical proteasomal degradation pathway, a non-canonical ubiquitin-dependent pathway maintains normal Glc7 localization and activity.This article has an associated First Person interview with the first author of the paper.