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.
Peroxisomes are organelles that perform beta-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their disfunction. Among disease-causing mutant genes are those required for protein transport into the peroxisome. The peroxisomal protein import machinery, also shared with chloroplasts, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes and current models postulate a large pore formed by transmembrane proteins. To date, however, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes membrane proteins Pex13 and Pex14 and cargo protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid-liquid phase separation (LLPS) with Pex5-cargo. Intrinsically disordered regions (IDR) in Pex13 and Pex5 resemble those found in nuclear pore complex (NPC) proteins. Cargo transport into peroxisomes depends on the number but not patterns of aromatic residues in these IDRs, consistent with their roles as stickers in associative polymer models of LLPS. Finally, imaging Fluorescence Cross-Correlation Spectroscopy (iFCCS) shows that the transport of cargo correlates with transient focusing of GFP-Pex13/14 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 results suggest a model in which LLPS of Pex5-cargo with Pex13/14 results in transient protein transport channels.
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.
Kozhikode district of North Kerala, India witnessed an outbreak of Nipah virus (NiV) in the month of May 2018. Two adjacent districts were affected leaving 17 patients dead out of the 19 confirmed. United Nations and WHO lauded the expeditious response of the state’s health system in the diagnosis and containment of the outbreak which was unprecedented. The authors being in the contact tracing and surveillance operation district team, had kept a record of timeline of events and actions at the state level, compiled the news clippings and tracked events. In the absence of an end‑of‑epidemic report for reference, these records served as a valuable tool for the present review. We used the Management science for health frame work tool (MSH framework) to evaluate the district and state coordinated actions which helped in curbing the outbreak. Though NiV outbreak in South India (2018) had similar epidemiological features to previous disease outbreaks, it stands out as the one to be detected and contained in a short span of time. As health personnel working in the government medical college of an affected district and directly involved in contact tracing operations and containment measures, exploring and sharing, what worked and how, in the context of multidisciplinary response and recovery attempts of the outbreak in the state may be beneficial to public health personnel and policy makers. This management framework may be replicated in the national and international context, particularly in South East Asian region under threat of emerging viral infections like COVID-19, lacking specific epidemic management frameworks for outbreak response and containment.
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