Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

CDK and MAPK Synergistically Regulate Signaling Dynamics via a Shared Multi-site Phosphorylation Region on the Scaffold Protein Ste5.

We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell-cycle stages. Surprisingly, in S phase, where Ste5 recruitment should be blocked, we observed an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK modify shared sites, which are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Such collaborative phosphorylation can broaden regulatory inputs and diversify output dynamics of signaling pathways.

Meiotic chromosome homology search involves modifications of the nuclear envelope protein Matefin/SUN-1.

Genome haploidization during meiosis depends on recognition and association of parental homologous chromosomes. The C. elegans SUN/KASH domain proteins Matefin/SUN-1 and ZYG-12 have a conserved role in this process. They bridge the nuclear envelope, connecting the cytoplasm and the nucleoplasm to transmit forces that allow chromosome movement and homolog pairing and prevent nonhomologous synapsis. Here, we show that Matefin/SUN-1 forms rapidly moving aggregates at putative chromosomal attachment sites in the meiotic transition zone (TZ). We analyzed requirements for aggregate formation and identified multiple phosphotarget residues in the nucleoplasmic domain of Matefin/SUN-1. These CHK-2 dependent phosphorylations occur in leptotene/zygotene, diminish during pachytene and are involved in pairing. Mimicking phosphorylation causes an extended TZ and univalents at diakinesis. Our data suggest that the properties of the nuclear envelope are altered during the time window when homologs are sorted and Matefin/SUN-1 aggregates form, thereby controling the movement, homologous pairing and interhomolog recombination of chromosomes.

A phosphatase-centric mechanism drives stress signaling response.

Changing environmental cues lead to the adjustment of cellular physiology by phosphorylation signaling networks that typically center around kinases as active effectors and phosphatases as antagonistic elements. Here, we report a signaling mechanism that reverses this principle. Using the hyperosmotic stress response in Saccharomyces cerevisiae as a model system, we find that a phosphatase-driven mechanism causes induction of phosphorylation. The key activating step that triggers this phospho-proteomic response is the Endosulfine-mediated inhibition of protein phosphatase 2A-Cdc55 (PP2ACdc55 ), while we do not observe concurrent kinase activation. In fact, many of the stress-induced phosphorylation sites appear to be direct substrates of the phosphatase, rendering PP2ACdc55 the main downstream effector of a signaling response that operates in parallel and independent of the well-established kinase-centric stress signaling pathways. This response affects multiple cellular processes and is required for stress survival. Our results demonstrate how a phosphatase can assume the role of active downstream effectors during signaling and allow re-evaluating the impact of phosphatases on shaping the phosphorylome.

Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase.

Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes.

MAPK Hog1 closes the S. cerevisiae glycerol channel Fps1 by phosphorylating and displacing its positive regulators.

The aquaglyceroprin Fps1 is responsible for glycerol transport in yeast in response to changes in extracellular osmolarity. Control of Fps1 channel activity in response to hyperosmotic shock involves a redundant pair of regulators, Rgc1 (regulator of the glycerol channel 1) and Rgc2, and the MAPK Hog1 (high-osmolarity glycerol response 1). However, the mechanism by which these factors influence channel activity is unknown. We show that Rgc2 maintains Fps1 in the open channel state in the absence of osmotic stress by binding to its C-terminal cytoplasmic domain. This interaction involves a tripartite pleckstrin homology (PH) domain within Rgc2 and a partial PH domain within Fps1. Activation of Hog1 in response to hyperosmotic shock induces the rapid eviction of Rgc2 from Fps1 and consequent channel closure. Hog1 was recruited to the N-terminal cytoplasmic domain of Fps1, which it uses as a platform from which to multiply phosphorylate Rgc2. Thus, these results reveal the mechanism by which Hog1 regulates Fps1 in response to hyperosmotic shock.

Novel interconnections of HOG signaling revealed by combined use of two proteomic software packages.

Modern quantitative mass spectrometry (MS)-based proteomics enables researchers to unravel signaling networks by monitoring proteome-wide cellular responses to different stimuli. MS-based analysis of signaling systems usually requires an integration of multiple quantitative MS experiments, which remains challenging, given that the overlap between these datasets is not necessarily comprehensive. In a previous study we analyzed the impact of the yeast mitogen-activated protein kinase (MAPK) Hog1 on the hyperosmotic stress-affected phosphorylome. Using a combination of a series of hyperosmotic stress and kinase inhibition experiments, we identified a broad range of direct and indirect substrates of the MAPK. Here we re-evaluate this extensive MS dataset and demonstrate that a combined analysis based on two software packages, MaxQuant and Proteome Discoverer, increases the coverage of Hog1-target proteins by 30%. Using protein-protein proximity assays we show that the majority of new targets gained by this analysis are indeed Hog1-interactors. Additionally, kinetic profiles indicate differential trends of Hog1-dependent versus Hog1-independent phosphorylation sites. Our findings highlight a previously unrecognized interconnection between Hog1 signaling and the RAM signaling network, as well as sphingolipid homeostasis.

Controlling gene expression in response to stress.

Acute stress puts cells at risk, and rapid adaptation is crucial for maximizing cell survival. Cellular adaptation mechanisms include modification of certain aspects of cell physiology, such as the induction of efficient changes in the gene expression programmes by intracellular signalling networks. Recent studies using genome-wide approaches as well as single-cell transcription measurements, in combination with classical genetics, have shown that rapid and specific activation of gene expression can be accomplished by several different strategies. This article discusses how organisms can achieve generic and specific responses to different stresses by regulating gene expression at multiple stages of mRNA biogenesis from chromatin structure to transcription, mRNA stability and translation.

Sfp1 interaction with TORC1 and Mrs6 reveals feedback regulation on TOR signaling.

Ribosome biogenesis drives cell growth, and the large transcriptional output underlying this process is tightly regulated. The Target of Rapamycin (TOR) kinase is part of a highly conserved signaling pathway linking nutritional and stress signals to regulation of ribosomal protein (RP) and ribosome biogenesis (Ribi) gene transcription. In Saccharomyces cerevisiae, one of the downstream effectors of TOR is Sfp1, a transcriptional activator that regulates both RP and Ribi genes. Here, we report that Sfp1 interacts directly with TOR complex 1 (TORC1) in a rapamycin-regulated manner, and that phosphorylation of Sfp1 by this kinase complex regulates its function. Sfp1, in turn, negatively regulates TORC1 phosphorylation of Sch9, another key TORC1 target that acts in parallel with Sfp1, revealing a feedback mechanism controlling the activity of these proteins. Finally, we show that the Sfp1-interacting protein Mrs6, a Rab escort protein involved in membrane trafficking, regulates both Sfp1 nuclear localization and TORC1 signaling.

H3K4 monomethylation dictates nucleosome dynamics and chromatin remodeling at stress-responsive genes.

Chromatin remodeling is essential for proper adaptation to extracellular stimuli. The p38-related Hog1 SAPK is an important regulator of transcription that mediates chromatin remodeling upon stress. Hog1 targets the RSC chromatin remodeling complex to stress-responsive genes and rsc deficient cells display reduced induction of gene expression. Here we show that the absence of H3K4 methylation, either achieved by deletion of the SET1 methyltransferase or by amino acid substitution of H3K4, bypasses the requirement of RSC for stress-responsive gene expression. Monomethylation of H3K4 is specifically inhibiting RSC-independent chromatin remodeling and thus, it prevents osmostress-induced gene expression. The absence of H3K4 monomethylation permits that the association of alternative remodelers with stress-responsive genes and the Swr1 complex (SWR-C) is instrumental in the induction of gene expression upon stress. Accordingly, the absence of SWR-C or histone H2A.Z results in compromised chromatin remodeling and impaired gene expression in the absence of RSC and H3K4 methylation. These results indicate that expression of stress-responsive genes is controlled by two remodeling mechanisms: RSC in the presence of monomethylated H3K4, and SWR-C in the absence of H3K4 monomethylation. Our findings point to a novel role for H3K4 monomethylation in dictating the specificity of chromatin remodeling, adding an extra layer of regulation to the transcriptional stress response.

Msb2 is a Ste11 membrane concentrator required for full activation of the HOG pathway.

The high osmolarity glycerol (HOG) pathway, composed of membrane-associated osmosensors, adaptor proteins and core signaling kinases, is essential for the survival of yeast cells under hyper-osmotic stress. Here, we studied how the MAPKKK Ste11 might change its protein interaction profile during acute stress exposure, with an emphasis on the sensory system of the so-called Sho1/Msb2 signaling branch. To characterize the transience of protein-protein interactions we utilized a recently described enzymatic in vivo protein proximity assay (M-track). Accordingly, interaction signals between Ste11 and many of its signaling partners can already be detected even under basal conditions. In most cases these signals increase after stress induction. All the interactions are completely dependent on the function of the Ste11-adaptor protein Ste50. Moreover, the presence of either Msb2 or Hkr1 is necessary for observing the interaction between Ste11 and scaffolding factors such as Sho1 and Pbs2. Additional assays suggest that Msb2 is not only in close proximity to Ste11 but might function as an individual Ste11 concentrator at the plasma membrane. Our results confirm the existence of negative feedback systems targeting the protein levels of Ste11 and Msb2 and also hint at changes in the dissociation rates of intermediate signaling complexes.

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy.

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.

A reversible gene trap collection empowers haploid genetics in human cells.

Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-ß, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.

Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19.

Autophagy is the major pathway for the delivery of cytoplasmic material to the vacuole or lysosome. Selective autophagy is mediated by cargo receptors, which link the cargo to the scaffold protein Atg11 and to Atg8 family proteins on the forming autophagosomal membrane. We show that the essential kinase Hrr25 activates the cargo receptor Atg19 by phosphorylation, which is required to link cargo to the Atg11 scaffold, allowing selective autophagy to proceed. We also find that the Atg34 cargo receptor is regulated in a similar manner, suggesting a conserved mechanism.

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

Conserved features and major differences in the outer membrane protein composition of chlamydiae.

Chlamydiae are a highly successful group of obligate intracellular bacteria infecting a variety of eukaryotic hosts. Outer membrane proteins involved in attachment to and uptake into host cells, and cross-linking of these proteins via disulfide bonds are key features of the biphasic chlamydial developmental cycle. In this study, we used a consensus approach to predict outer membrane proteins in the genomes of members of three chlamydial families. By analysing outer membrane protein fractions of purified chlamydiae with highly sensitive mass spectrometry, we show that the protein composition differs strongly between these organisms. Large numbers of major outer membrane protein-like proteins are present at high abundance in the outer membrane of Simkania negevensis and Waddlia chondrophila, whereas yet uncharacterized putative porins dominate in Parachlamydia acanthamoebae. Simkania represents the first case of a chlamydia completely lacking stabilizing cysteine-rich proteins in its outer membrane. In agreement with this, and in contrast to Parachlamydia and Waddlia, the cellular integrity of Simkania is not impaired by conditions that reduce disulfide bonds of these proteins. The observed differences in the protein composition of the outer membrane among members of divergent chlamydial families suggest different stabilities of these organisms in the environment, probably due to adaption to different niches or transmission routes.

M-Track: detecting short-lived protein-protein interactions in vivo.

We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.

An in vivo detection system for transient and low-abundant protein interactions and their kinetics in budding yeast.

Methylation tracking (M-Track) is a protein-proximity assay in Saccharomyces cerevisiae, allowing the detection of transient protein-protein interactions in living cells. The bait protein is fused to a histone lysine methyl transferase and the prey protein to a methylation acceptor peptide derived from histone 3. Upon interaction, the histone 3 fragment is stably methylated on lysine 9 and can be detected by methylation-specific antibodies. Since methylation marking is irreversible in budding yeast and only takes place in living cells, the occurrence of artifacts during cell lysate preparation is greatly reduced, leading to a more accurate representation of native interactions. So far, this method has been limited to highly abundant or overexpressed proteins. However, many proteins of interest are low-abundant, and overexpression of proteins may interfere with their function, leading to an artificial situation. Here we report the generation of a toolbox including a novel cleavage-enrichment system for the analysis of very low-abundant proteins at their native expression levels. In addition, we developed a system for the parallel analysis of two prey proteins in a single cell, as well as an inducible methylation system. The inducible system allows precise control over the time during which the interaction is detected and can be used to determine interaction kinetics. Furthermore, we generated a set of constructs facilitating the cloning-free genomic tagging of proteins at their endogenous locus by homologous recombination, and their expression from centromeric plasmids.

The Hog1 stress-activated protein kinase targets nucleoporins to control mRNA export upon stress.

The control of mRNA biogenesis is exerted at several steps. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress by acting during transcriptional initiation and elongation. Here, we genetically show that an intact nuclear pore complex is important for cell survival and maximal expression of stress-responsive genes. The Hog1 SAPK associates with nuclear pore complex components and directly phosphorylates the Nup1, Nup2, and Nup60 components of the inner nuclear basket. Mutation of those factors resulted in a deficient export of stress-responsive genes upon stress. Association of Nup1, Nup2, and Nup60 to stress-responsive promoters occurs upon stress depending on Hog1 activity. Accordingly, STL1 gene territory is maintained at the nuclear periphery upon osmostress in a Hog1-dependent manner. Cells containing non-phosphorylatable mutants in Nup1 or Nup2 display reduced expression of stress-responsive genes. Together, proper mRNA biogenesis of stress-responsive genes requires of the coordinate action of synthesis and export machineries by the Hog1 SAPK.

Studying the fragmentation behavior of peptides with arginine phosphorylation and its influence on phospho-site localization.

Phospho-proteomic studies opened a broad view onto the main mechanisms of regulating cellular processes. Our recent discovery of a protein arginine kinase and its target in bacteria added a previously undescribed type of phosphorylation to control protein activity. Several challenges arise from large in vivo studies of this and other types of phosphorylations. The main factors impeding correct localization are low spectral quality, neutral loss of phosphoric acid, and gas-phase rearrangements, which have recently been described for phospho-serine, -threonine, and -tyrosine. Studies on histidine-phosphorylated peptides, a nitrogen-bound phosphorylation, also reported loss of phosphoric acid upon collision-induced dissociation. We were interested in studying the behaviour of arginine phosphorylation under different fragmentation conditions and its influence on site localization. First, we determined the percentage of false localizations obtained by three different search engines and a software tool dedicated for phospho-site determination. Next, we demonstrate that application of collisional activation for analysis of arginine-phosphorylated peptides leads to extensive elimination of phosphoric acid and increases the numbers of false localizations, while the modification is maintained on the arginine side chain upon electron-transfer dissociation. Furthermore, we also observed a rearrangement of the phosphorylation onto serine and glutamic acid side chains upon collisional activation.