Synthesis of biologically active proteins as L6KD-SUMO fusions forming inclusion bodies in Escherichia coli.

A new platform has been developed to facilitate the production of biologically active proteins and peptides in Escherichia coli. The platform includes an N-terminal self-associating L6 KD peptide fused to the SUMO protein (small ubiquitin-like protein modifier) from the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. The target proteins are fused at the C termini of the L6 KD-SUMO fusions, and the resulting three-component fusion proteins are synthesized and self-assembled in E. coli into so-called active inclusion bodies (AIBs). In vivo, the L6 KD-SUMO platform facilitates the correct folding of the target proteins and directs them into AIBs, greatly simplifying their purification. In vitro, the platform facilitates the effective separation of AIBs by centrifugation and subsequent target protein release using SUMO-specific protease. The properties of the AIBs were determined using five proteins with different sizes, folding efficiencies, quaternary structure, and disulfide modifications. Electron microscopy shows that AIBs are synthesized in the form of complex fibrillar structures resembling "loofah sponges" with unusually thick filaments. The obtained results indicate that the new platform has promising features and could be developed to facilitate the synthesis and purification of target proteins and protein complexes without the use of renaturation.

Vaccine building 'kit': combining peptide bricks to elicit a desired immune response without adding an adjuvant.

Protein nanoparticles (NPs) can be used as vaccine platforms for target antigen presentation. Aim: To conduct a proof-of-concept study to demonstrate that an effective NP platform can be built based on a short self-assembling peptide (SAP) rather than a large self-assembling protein. Materials & methods: SUMO-based protein fusions (SFs) containing an N-terminal SAP and a C-terminal antigen were designed, expressed in Escherichia coli and purified. The structure was investigated by electron microscopy. The antibody response was tested in mice after two adjuvant-free immunizations. Results: Renatured SFs form fiber-like NPs with the antigen exposed on the surface and induce a significant antibody response with a remarkably high target-to-platform ratio. Conclusion: The platform is effective and has considerable potential for modification toward various applications, including vaccine development.

We aimed to extend the arsenal of protein platforms used for vaccine development. To this end, in this proof-of-concept study we constructed new self-assembling fusion proteins consisting of three modules. Module 1 is responsible for the self-assembly, while modules 2 and 3 are responsible for the immune response. Modules 1 and 2 form the platform, while module 3 represents the target antigen exposed on the surface of the self-assembled nanoparticles. After conventional biosynthesis in Escherichia coli, the proteins undergo efficient self-assembly during purification, and the resulting nanoparticles elicit a strong immune response without using an enhancing agent (adjuvant). The simple modular design and a high target-to-platform ratio of the immune response make our system a promising approach for practical applications, including vaccine development.

A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6.

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson's disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.

Mitochondrial Voltage-Dependent Anion Channel Protein Por1 Positively Regulates the Nuclear Localization of Saccharomyces cerevisiae AMP-Activated Protein Kinase.

Snf1 protein kinase of the yeast Saccharomyces cerevisiae is a member of the highly conserved eukaryotic AMP-activated protein kinase (AMPK) family, which is involved in regulating responses to energy limitation. Under conditions of carbon/energy stress, such as during glucose depletion, Snf1 is catalytically activated and enriched in the nucleus to regulate transcription. Snf1 catalytic activation requires phosphorylation of its conserved activation loop threonine (Thr210) by upstream kinases. Catalytic activation is also a prerequisite for Snf1's subsequent nuclear enrichment, a process that is mediated by Gal83, one of three alternate ß-subunits of the Snf1 kinase complex. We previously reported that the mitochondrial voltage-dependent anion channel (VDAC) proteins Por1 and Por2 play redundant roles in promoting Snf1 catalytic activation by Thr210 phosphorylation. Here, we show that the por1Δ mutation alone, which by itself does not affect Snf1 Thr210 phosphorylation, causes defects in Snf1 and Gal83 nuclear enrichment and Snf1's ability to stimulate transcription. We present evidence that Por1 promotes Snf1 nuclear enrichment by promoting the nuclear enrichment of Gal83. Overexpression of Por2, which is not believed to have channel activity, can suppress the localization and transcription activation defects of the por1Δ mutant, suggesting that the regulatory role played by Por1 is separable from its channel function. Thus, our findings expand the positive roles of the yeast VDACs in carbon/energy stress signaling upstream of Snf1. Since AMPK/Snf1 and VDAC proteins are conserved in evolution, our findings in yeast may have implications for AMPK regulation in other eukaryotes, including humans. IMPORTANCE AMP-activated protein kinases (AMPKs) sense energy limitation and regulate transcription and metabolism in eukaryotes from yeast to humans. In mammals, AMPK responds to increased AMP-to-ATP or ADP-to-ATP ratios and is implicated in diabetes, heart disease, and cancer. Mitochondria produce ATP and are generally thought to downregulate AMPK. Indeed, some antidiabetic drugs activate AMPK by affecting mitochondrial respiration. ATP release from mitochondria is mediated by evolutionarily conserved proteins known as voltage-dependent anion channels (VDACs). One would therefore expect VDACs to serve as negative regulators of AMPK. However, our experiments in yeast reveal the existence of an opposite relationship. We previously showed that Saccharomyces cerevisiae VDACs Por1 and Por2 positively regulate AMPK/Snf1 catalytic activation. Here, we show that Por1 also plays an important role in promoting AMPK/Snf1 nuclear localization. Our counterintuitive findings could inform research in areas ranging from diabetes to cancer to fungal pathogenesis.

Quaternary structure of the yeast pheromone receptor Ste2 in living cells.

Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8·10-6 to 3.5·10-5molecules/nm2), and a mixture of tetramers and octamers at expression levels of 25-100 molecules per pixel (3.5·10-5 to 1.4·10-4molecules/nm2). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a, and binds the pheromone α-factor secreted by cells of the mating type α. Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Springing into Action: Reg2 Negatively Regulates Snf1 Protein Kinase and Facilitates Recovery from Prolonged Glucose Starvation in Saccharomyces cerevisiae.

UNLABELLED: Glucose is the preferred carbon source for the yeast Saccharomyces cerevisiae Glucose limitation activates Snf1 protein kinase, a key regulator of energy homeostasis that promotes utilization of alternative carbon sources and enforces energy conservation. Snf1 activation requires phosphorylation of its T-loop threonine (Thr210) by upstream kinases. When glucose is abundant, Snf1 is inhibited by Thr210 dephosphorylation. This involves the function of the type 1 protein phosphatase Glc7, which is targeted to Snf1 by a regulatory subunit, Reg1. The reg1 mutation causes increased Snf1 activity and mimics various aspects of glucose limitation, including slower growth. Reg2 is another Glc7 regulatory subunit encoded by a paralogous gene, REG2 Previous evidence indicated that the reg2 mutation exacerbates the Snf1-dependent slow-growth phenotype caused by reg1, suggesting a link between Reg2 and Snf1. Here, we explore this link in more detail and present evidence that Reg2 contributes to Snf1 Thr210 dephosphorylation. Consistent with this role, Reg2 interacts with wild-type Snf1 but not with nonphosphorylatable Snf1-T210A. Reg2 accumulation increases in a Snf1-dependent manner during prolonged glucose deprivation, and glucose-starved cells lacking Reg2 exhibit delayed Snf1 Thr210 dephosphorylation and slower growth recovery upon glucose replenishment. Accordingly, cells lacking Reg2 are outcompeted by wild-type cells in the course of several glucose starvation/replenishment cycles. Collectively, our results support a model in which Reg2-Glc7 contributes to the negative control of Snf1 in response to glucose refeeding after prolonged starvation. The competitive growth advantage provided by Reg2 underscores the evolutionary significance of this paralog for S. cerevisiae IMPORTANCE: The ability of microorganisms to respond to stress is essential for their survival. However, rapid recovery from stress could be equally crucial in competitive environments. Therefore, a wise stress response program should prepare cells for quick recovery upon reexposure to favorable conditions. Glucose is the preferred carbon source for the yeast S. cerevisiae Glucose depletion activates the stress response protein kinase Snf1, which functions to limit energy-consuming processes, such as growth. We show that prolonged glucose deprivation also leads to Snf1-dependent accumulation of Reg2 and that this protein helps to inhibit Snf1 and to accelerate growth recovery upon glucose replenishment. Cells lacking Reg2 are readily outcompeted by wild-type cells during glucose depletion/replenishment cycles. Thus, while prolonged glucose deprivation might seem to put yeast cells "on their knees," concomitant accumulation of Reg2 helps configure the cells into a "sprinter's crouch start position" to spring into action once glucose becomes available.

Development and experimental testing of an optical micro-spectroscopic technique incorporating true line-scan excitation.

Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects.

Mitochondrial porin Por1 and its homolog Por2 contribute to the positive control of Snf1 protein kinase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Snf1 is a member of the conserved Snf1/AMP-activated protein kinase (Snf1/AMPK) family involved in regulating responses to energy limitation, which is detected by mechanisms that include sensing adenine nucleotides. Mitochondrial voltage-dependent anion channel (VDAC) proteins, also known as mitochondrial porins, are conserved in eukaryotes from yeast to humans and play key roles in mediating mitochondrial outer membrane permeability to small metabolites, including ATP, ADP, and AMP. We previously recovered the yeast mitochondrial porin Por1 (yVDAC1) from a two-hybrid screen for Snf1-interacting proteins. Here, we present evidence that Snf1 interacts with Por1 and its homolog Por2 (yVDAC2). Cells lacking Por1 and Por2, but not respiratory-deficient rho(0) cells lacking the mitochondrial genome, exhibit reduced Snf1 activation loop phosphorylation in response to glucose limitation. Thus, Por1 and Por2 contribute to the positive control of Snf1 protein kinase. Physical proximity to the VDAC proteins and mitochondrial surface could facilitate Snf1's ability to sense energy limitation.

Protein kinase A contributes to the negative control of Snf1 protein kinase in Saccharomyces cerevisiae.

Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.

Roles of the Snf1-activating kinases during nitrogen limitation and pseudohyphal differentiation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Snf1 protein kinase is important for growth on carbon sources that are less preferred than glucose. When glucose becomes limiting, Snf1 undergoes catalytic activation, which requires phosphorylation of its T-loop threonine (Thr210). Thr210 phosphorylation can be performed by any of three Snf1-activating kinases: Sak1, Tos3, and Elm1. These kinases are redundant in that all three must be eliminated to confer snf1Delta-like growth defects on nonpreferred carbon sources. We previously showed that in addition to glucose signaling, Snf1 also participates in nitrogen signaling and is required for diploid pseudohyphal differentiation, a filamentous-growth response to nitrogen limitation. Here, we addressed the roles of the Snf1-activating kinases in this process. Loss of Sak1 caused a defect in pseudohyphal differentiation, whereas Tos3 and Elm1 were dispensable. Sak1 was also required for increased Thr210 phosphorylation of Snf1 under nitrogen-limiting conditions. Expression of a catalytically hyperactive version of Snf1 restored pseudohyphal differentiation in the sak1Delta/sak1Delta mutant. Thus, while the Snf1-activating kinases exhibit redundancy for growth on nonpreferred carbon sources, the loss of Sak1 alone produced a significant defect in a nitrogen-regulated phenotype, and this defect resulted from deficient Snf1 activation rather than from disruption of another pathway. Our results suggest that Sak1 is involved in nitrogen signaling upstream of Snf1.

Detection of endogenous Snf1 and its activation state: application to Saccharomyces and Candida species.

The stress-response Snf1 protein kinase of Saccharomyces cerevisiae serves as a powerful model for studies of the eukaryotic Snf1/AMP-activated protein kinase (AMPK) family. Central to studies of Snf1 are methods that determine its activation state under various physiological and genetic conditions. Here, we have developed a convenient and sensitive method for immunoblot analysis of endogenous yeast Snf1 and its activation-loop threonine (Thr210) phosphorylation. The method employs readily obtainable reagents and yields results that faithfully reflect the environmental and genetic conditions tested. Using our method, we have obtained evidence that Snf1 remains stress-regulated in reg1 Delta cells, revealing the existence of a Snf1 signalling mechanism(s) that is independent of Reg1-PP1 phosphatase. In addition to strains of common laboratory S. cerevisiae backgrounds, we have applied the method to two pathogenic Candida species, C. glabrata and C. albicans. We have detected proteins whose gel mobilities, immune properties and regulation patterns are consistent with those expected for the corresponding Snf1 homologues. Because Snf1 activation is a sensitive marker of several types of stress, including artifactual stresses associated with common cell harvesting and protein extraction procedures, the convenient and efficient protein extraction method described here should be advantageous for SDS-PAGE and immunoblot analyses of stress-regulated and other proteins from various yeast species.

Nitrogen availability and TOR regulate the Snf1 protein kinase in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the Snf1 protein kinase of the Snf1/AMP-activated protein kinase (AMPK) family regulates a wide range of responses to stress caused by glucose deprivation. The stress signal is relayed via upregulation of Snf1, which depends on phosphorylation of its activation loop Thr210 residue by upstream kinases. Although Snf1 is also required for coping with various stresses unrelated to glucose deprivation, some evidence suggests a role for low-level basal activity of unphosphorylated Snf1, rather than a specific signaling function. We previously found that Snf1 is required for diploid pseudohyphal differentiation, a developmental response to nitrogen limitation. Here, we present evidence that Snf1 is directly involved in nitrogen signaling. First, genetic analyses suggest that pseudohyphal differentiation depends on the stimulatory phosphorylation of Snf1 at Thr210. Second, immunochemical data indicate that nitrogen limitation improves Thr210 phosphorylation. Analyses of pseudohyphal differentiation in cells with catalytically inactive and hyperactive Snf1 support the role of Snf1 activity. Finally, we show that Snf1 is negatively regulated by the rapamycin-sensitive TOR kinase which plays essential roles in signaling nitrogen and amino acid availability. This and other evidence implicate Snf1 in the integration of signals regarding nitrogen and carbon stress. TOR and Snf1/AMPK are highly conserved in evolution, and their novel functional interaction in yeast suggests similar mechanisms in other eukaryotes.

Std1p (Msn3p) positively regulates the Snf1 kinase in Saccharomyces cerevisiae.

The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p. Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1. Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors. Here we address the relationship between Std1p and the Snf1 kinase. Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains. Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex. Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays. These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.

Snf1 kinases with different beta-subunit isoforms play distinct roles in regulating haploid invasive growth.

The Snf1 protein kinase of Saccharomyces cerevisiae has been shown to have a role in regulating haploid invasive growth in response to glucose depletion. Cells contain three forms of the Snf1 kinase, each with a different beta-subunit isoform, either Gal83, Sip1, or Sip2. We present evidence that different Snf1 kinases play distinct roles in two aspects of invasive growth, namely, adherence to the agar substrate and filamentation. The Snf1-Gal83 form of the kinase is required for adherence, whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation. Genetic evidence indicates that Snf1-Gal83 affects adherence by antagonizing Nrg1- and Nrg2-mediated repression of the FLO11 flocculin and adhesin gene. In contrast, the mechanism(s) by which Snf1-Gal83 and Snf1-Sip2 affect filamentation is independent of FLO11. Thus, the Snf1 kinase regulates invasive growth by at least two distinct mechanisms.

Snf1 protein kinase and the repressors Nrg1 and Nrg2 regulate FLO11, haploid invasive growth, and diploid pseudohyphal differentiation.

The Snf1 protein kinase of Saccharomyces cerevisiae is important for many cellular responses to glucose limitation, including haploid invasive growth. We show here that Snf1 regulates transcription of FLO11, which encodes a cell surface glycoprotein required for invasive growth. We further show that Nrg1 and Nrg2, two repressor proteins that interact with Snf1, function as negative regulators of invasive growth and as repressors of FLO11. We also examined the role of Snf1, Nrg1, and Nrg2 in two other Flo11-dependent processes. Mutations affected the initiation of biofilm formation, which is glucose sensitive, but also affected diploid pseudohyphal differentiation, thereby unexpectedly implicating Snf1 in a response to nitrogen limitation. Deletion of the NRG1 and NRG2 genes suppressed the defects of a snf1 mutant in all of these processes. These findings suggest a model in which the Snf1 kinase positively regulates Flo11-dependent developmental events by antagonizing Nrg-mediated repression of the FLO11 gene.