RESUMO
Autophagy is a conserved process that recycles cellular contents to promote survival. Although nitrogen limitation is the canonical inducer of autophagy, recent studies have revealed several other nutrients important to this process. In this study, we used a quantitative, high-throughput assay to identify potassium starvation as a new and potent inducer of autophagy in the yeast Saccharomyces cerevisiae We found that potassium-dependent autophagy requires the core pathway kinases Atg1, Atg5, and Vps34, and other components of the phosphatidylinositol 3-kinase complex. Transmission EM revealed abundant autophagosome formation in response to both stimuli. RNA-Seq indicated distinct transcriptional responses: nitrogen affects transport of ions such as copper, whereas potassium targets the organization of other cellular components. Thus, nitrogen and potassium share the ability to influence molecular supply and demand but do so in different ways. Both inputs promote catabolism through bulk autophagy, but result in distinct mechanisms of cellular remodeling and synthesis.
Assuntos
Autofagia , Potássio/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein ßγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gßγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.
Assuntos
Classe III de Fosfatidilinositol 3-Quinases/genética , Proteínas F-Box/genética , Genes Fúngicos Tipo Acasalamento/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Ciclo Celular/genética , Endossomos/genética , Proteínas F-Box/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Morfogênese/genética , Feromônios/genética , Feromônios/metabolismo , Saccharomyces cerevisiae/fisiologia , Deleção de Sequência/genética , Transdução de Sinais , Transcrição Gênica , Vacúolos/genética , Vacúolos/metabolismo , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Antimicrobial peptides (AMPs) cause bacterial membrane permeabilization and ultimately cell death at low µM concentrations. The membrane permeabilization action of a moth derived AMP Cecropin A on E. coli cells in exponential growth (mid-log phase) is well studied. At 1× MIC concentration, Cecropin A penetrates the lipopolysaccharide (LPS) barrier and causes outer membrane (OM) and cytoplasmic membrane (CM) permeabilization. For non-septating cells, permeabilization of both membranes begins at one pole. For septating cells, OM permeabilization begins at the septal region and CM permeabilization begins at one pole. However, in nature bacteria are frequently found in nutrient-starved conditions. Here we extend our single-cell microscopy assays to the attack of Cecropin A on E. coli cells in early stationary phase. Stationary phase E. coli is much more resistant to membrane permeabilization by Cecropin A than mid-log phase E. coli. A tenfold higher concentration of Cecropin A is required to observe CM permeabilization in the majority of stationary phase cells, and even then permeabilization proceeds more slowly. In addition, the spatial pattern of initial CM permeabilization changes from localized at one pole to global. Studies of lipid mutant strains suggest that a sufficient localized concentration of the anionic phospholipid phosphatidylglycerol (PG) guides the position of initial attack of the cationic AMP Cecropin A on the CM.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Escherichia coli/crescimento & desenvolvimento , Lipopolissacarídeos/metabolismo , Membranas/metabolismo , Microscopia de Fluorescência/métodosRESUMO
In Escherichia coli, FtsLB plays a central role in the initiation of cell division, possibly transducing a signal that will eventually lead to the activation of peptidoglycan remodeling at the forming septum. The molecular mechanisms by which FtsLB operates in the divisome, however, are not understood. Here, we present a structural analysis of the FtsLB complex, performed with biophysical, computational, and in vivo methods, that establishes the organization of the transmembrane region and proximal coiled coil of the complex. FRET analysis in vitro is consistent with formation of a tetramer composed of two FtsL and two FtsB subunits. We predicted subunit contacts through co-evolutionary analysis and used them to compute a structural model of the complex. The transmembrane region of FtsLB is stabilized by hydrophobic packing and by a complex network of hydrogen bonds. The coiled coil domain probably terminates near the critical constriction control domain, which might correspond to a structural transition. The presence of strongly polar amino acids within the core of the tetrameric coiled coil suggests that the coil may split into two independent FtsQ-binding domains. The helix of FtsB is interrupted between the transmembrane and coiled coil regions by a flexible Gly-rich linker. Conversely, the data suggest that FtsL forms an uninterrupted helix across the two regions and that the integrity of this helix is indispensable for the function of the complex. The FtsL helix is thus a candidate for acting as a potential mechanical connection to communicate conformational changes between periplasmic, membrane, and cytoplasmic regions.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Periplasma/química , Periplasma/genética , Periplasma/metabolismo , Estrutura Secundária de ProteínaRESUMO
Deeper understanding of the bacteriostatic and bactericidal mechanisms of antimicrobial peptides (AMPs) should help in the design of new antibacterial agents. Over several decades, a variety of biochemical assays have been applied to bulk bacterial cultures. While some of these bulk assays provide time resolution of the order of 1min, they do not capture faster mechanistic events. Nor can they provide subcellular spatial information or discern cell-to-cell heterogeneity within the bacterial population. Single-cell, time-resolved imaging assays bring a completely new spatiotemporal dimension to AMP mechanistic studies. We review recent work that provides new insights into the timing, sequence, and spatial distribution of AMP-induced effects on bacterial cells.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/citologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Microscopia de FluorescênciaRESUMO
Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of "dead-cell stains" (SYTOX orange and SYTOX green) and "live-cell stains" (DRAQ5 and SYTO 61) and also 4',6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/citologia , Bacillus subtilis/efeitos dos fármacos , Nucléolo Celular/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Bacillus subtilis/química , Bacillus subtilis/crescimento & desenvolvimento , Nucléolo Celular/química , Escherichia coli/crescimento & desenvolvimento , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência , Coloração e Rotulagem , CatelicidinasRESUMO
Fluorescence microscopy enables detailed observation of the effects of the antimicrobial peptide Cecropin A on the outer membrane (OM) and cytoplasmic membrane (CM) of single E. coli cells with subsecond time resolution. Fluorescence from periplasmic GFP decays and cell growth halts when the OM is permeabilized. Fluorescence from the DNA stain Sytox Green rises when the CM is permeabilized and the stain enters the cytoplasm. The initial membrane disruptions are localized and stable. Septating cells are attacked earlier than nonseptating cells, and curved membrane surfaces are attacked in preference to cylindrical surfaces. Below a threshold bulk Cecropin A concentration, permeabilization is not observed over 30 min. Above this threshold, we observe a lag time of several minutes between Cecropin A addition and OM permeabilization and â¼30 s between OM and CM permeabilization. The long lag times and the existence of a threshold concentration for permeabilization suggest a nucleation mechanism. However, the roughly linear dependence of mean lag time on bulk peptide concentration is not easily reconciled with a nucleation step involving simultaneous insertion of multiple peptides into the bilayer. Monte Carlo simulations suggest that within seconds, the OM permeability becomes comparable to that of a pore of 100 nm diameter or of numerous small pores distributed over a similarly large area.