Biol. 11, 235 (2011)], which acts as a bona fide Wnt5a-responsive element. Importantly, this phospho-dependent degradation is essential for Wnt5a-Ror modulation of cell migration. Collectively, this work establishes a Wnt5a-Ror cell morphogenetic cascade involving Pdzrn3 phosphorylation and degradation.Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from Piriformospora indica (PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.While RNAs are known to misfold, the underlying molecular causes have been mainly studied in fragments of biologically relevant larger RNAs. (R,S)-3,5-DHPG molecular weight As these small RNAs are dominated by secondary structures, misfolding of these secondary structures remains the most-explored cause for global RNA misfolding. Conversely, how RNA chaperones function in a biological context to promote native folding beyond duplex annealing remains unknown. Here, in a combination of dimethylsulfate mutational profiling with sequencing (DMS-MaPseq), structural analyses, biochemical experiments, and yeast genetics, we show that three-helix junctions are prone to misfolding during assembly of the small ribosomal subunit in vivo. We identify ubiquitous roles for ribosome assembly factors in chaperoning their folding by preventing the formation of premature tertiary interactions, which otherwise kinetically trap misfolded junctions, thereby blocking further progress in the assembly cascade. While these protein chaperones act indirectly by binding the interaction partners of junctions, our analyses also suggest direct roles for small nucleolar RNAs (snoRNAs) in binding and chaperoning helical junctions during transcription. While these assembly factors do not utilize energy to ameliorate misfolding, our data demonstrate how their dissociation renders reversible folding steps irreversible, thereby driving native folding and assembly and setting up a timer that dictates the propensity of misfolded intermediates to escape quality control. Finally, the data demonstrate that RNA chaperones act locally on individual tertiary interactions, in contrast to protein chaperones, which globally unfold misfolded proteins.Maintaining homeostasis is a fundamental characteristic of living systems. In cells, this is contributed to by the assembly of biochemically distinct organelles, many of which are not membrane bound but form by the physical process of liquid-liquid phase separation (LLPS). By analogy with LLPS in binary solutions, cellular LLPS was hypothesized to contribute to homeostasis by facilitating "concentration buffering," which renders the local protein concentration within the organelle robust to global variations in the average cellular concentration (e.g., due to expression noise). Interestingly, concentration buffering was experimentally measured in vivo in a simple organelle with a single solute, while it was observed not to be obeyed in one with several solutes. Here, we formulate theoretically and solve analytically a physical model of LLPS in a ternary solution of two solutes (ϕ and ψ) that interact both homotypically (ϕ-ϕ attractions) and heterotypically (ϕ-ψ attractions). Our physical theory predicts how the coexisting concentrations in LLPS are related to expression noise and thus, generalizes the concept of concentration buffering to multicomponent systems. This allows us to reconcile the seemingly contradictory experimental observations. Furthermore, we predict that incremental changes of the homotypic and heterotypic interactions among the molecules that undergo LLPS, such as those that are caused by mutations in the genes encoding the proteins, may increase the efficiency of concentration buffering of a given system. Thus, we hypothesize that evolution may optimize concentration buffering as an efficient mechanism to maintain LLPS homeostasis and suggest experimental approaches to test this in different systems.Proteome-wide profiling of protein phosphorylation has been widely used to reveal the underlying mechanism of diverse cellular signaling events. Yet, characterizing subcellular phosphoproteome with high spatial-temporal resolution has remained challenging. Herein, we developed a subcellular-specific uncaging-assisted biotinylation and mapping of phosphoproteome (SubMAPP) strategy to monitor the phosphorylation dynamics of subcellular proteome in living cells and animals. Our method capitalizes on the genetically encoded bioorthogonal decaging strategy, which enables the rapid activation of subcellular localized proximity labeling biotin ligase through either light illumination or small-molecule triggers. By further adopting an integrated orthogonal pull-down strategy with quantitative mass spectrometry, SubMAPP allowed for the investigation of subcellular phosphoproteome dynamics, revealing the altered phosphorylation patterns of endoplasmic reticulum (ER) luminal proteins under ER stress. Finally, we further expanded the scope of the SubMAPP strategy to primary neuron culture and living mice.