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Nature Communications Jan 2016Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles...
Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1-kinase complex. However, the underlying molecular mechanism remains unclear. Here we reconstitute this early step in autophagy from purified components in vitro. We find that on assembly from its cytoplasmic subcomplexes, the Atg1-kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. The scaffolding protein Atg17 targets the Atg1-kinase complex to autophagic membranes by specifically recognizing the membrane protein Atg9. This interaction is inhibited by the two regulatory subunits Atg31 and Atg29. Engagement of the Atg1-Atg13 subcomplex restores the Atg9-binding and membrane-tethering activity of Atg17. Our data help to unravel the mechanism that controls Atg17-mediated tethering of Atg9-vesicles, providing the molecular basis to understand initiation of autophagosome-biogenesis.
Topics: Adaptor Proteins, Signal Transducing; Alkaline Phosphatase; Autophagy; Autophagy-Related Proteins; Carrier Proteins; Circular Dichroism; Cryoelectron Microscopy; Dynamic Light Scattering; Immunoprecipitation; In Vitro Techniques; Liposomes; Membrane Proteins; Microscopy, Confocal; Organelle Biogenesis; Protein Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Subcellular Fractions; Tandem Mass Spectrometry
PubMed: 26753620
DOI: 10.1038/ncomms10338 -
Proceedings of the National Academy of... Jul 1997The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins,...
The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1-1 mutation. We now describe a third suppressor of sec1-1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.
Topics: Biological Transport; Cloning, Molecular; Fungal Proteins; Gene Expression Regulation, Fungal; Membrane Proteins; Membrane Transport Proteins; Qa-SNARE Proteins; SEC Translocation Channels; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 9207091
DOI: 10.1073/pnas.94.14.7331 -
Cell Cycle (Georgetown, Tex.) Apr 2017
Topics: Cell Cycle Proteins; Nuclear Proteins; Phosphoprotein Phosphatases; Protein-Tyrosine Kinases; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 28272970
DOI: 10.1080/15384101.2017.1302229 -
Cell Mar 2003The cohesin complex is essential for sister chromatid cohesion during mitosis. Its Smc1 and Smc3 subunits are rod-shaped molecules with globular ABC-like ATPases at one... (Comparative Study)
Comparative Study
The cohesin complex is essential for sister chromatid cohesion during mitosis. Its Smc1 and Smc3 subunits are rod-shaped molecules with globular ABC-like ATPases at one end and dimerization domains at the other connected by long coiled coils. Smc1 and Smc3 associate to form V-shaped heterodimers. Their ATPase heads are thought to be bridged by a third subunit, Scc1, creating a huge triangular ring that could trap sister DNA molecules. We address here whether cohesin forms such rings in vivo. Proteolytic cleavage of Scc1 by separase at the onset of anaphase triggers its dissociation from chromosomes. We show that N- and C-terminal Scc1 cleavage fragments remain connected due to their association with different heads of a single Smc1/Smc3 heterodimer. Cleavage of the Smc3 coiled coil is sufficient to trigger cohesin release from chromosomes and loss of sister cohesion, consistent with a topological association with chromatin.
Topics: Amino Acid Sequence; Anaphase; Binding Sites; Cell Cycle Proteins; Chondroitin Sulfate Proteoglycans; Chromatids; Chromatin; Chromosomal Proteins, Non-Histone; Chromosomes, Fungal; Conserved Sequence; DNA-Binding Proteins; Dimerization; Fungal Proteins; Molecular Sequence Data; Nuclear Proteins; Phosphoproteins; Protein Structure, Tertiary; Protein Subunits; Saccharomyces cerevisiae Proteins; Schizosaccharomyces pombe Proteins; Sequence Homology, Amino Acid; Yeasts; Cohesins
PubMed: 12654244
DOI: 10.1016/s0092-8674(03)00162-4 -
PLoS Pathogens Jan 2018
Topics: Dimerization; Gene Deletion; Glutathione Peroxidase; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Heat-Shock Proteins; Models, Molecular; Molecular Chaperones; Peptide Fragments; Phosphoproteins; Prion Proteins; Prions; Protein Interaction Domains and Motifs; Protein Multimerization; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 29300791
DOI: 10.1371/journal.ppat.1006695 -
Genetics Mar 2002In meiotic prophase of many eukaryotic organisms, telomeres attach to the nuclear envelope and form a polarized configuration called the bouquet. Bouquet formation is...
In meiotic prophase of many eukaryotic organisms, telomeres attach to the nuclear envelope and form a polarized configuration called the bouquet. Bouquet formation is hypothesized to facilitate homologous chromosome pairing. In fission yeast, bouquet formation and telomere clustering occurs in karyogamy and persists throughout the horsetail stage. Here we report the isolation and characterization of six mutants from our screen for meiotic mutants. These mutants show defective telomere clustering as demonstrated by mislocalization of Swi6::GFP, a heterochromatin-binding protein, and Taz1p::GFP, a telomere-specific protein. These mutants define four complementation groups and are named dot1 to dot4-defective organization of telomeres. dot3 and dot4 are allelic to mat1-Mm and mei4, respectively. Immunolocalization of Sad1, a protein associated with the spindle pole body (SPB), in dot mutants showed an elevated frequency of multiple Sad1-nuclei signals relative to wild type. Many of these Sad1 foci were colocalized with Taz1::GFP. Impaired SPB structure and function were further demonstrated by failure of spore wall formation in dot1, by multiple Pcp1::GFP signals (an SPB component) in dot2, and by abnormal microtubule organizations during meiosis in dot mutants. The coincidence of impaired SPB functions with defective telomere clustering suggests a link between the SPB and the telomere cluster.
Topics: Cell Cycle Proteins; DNA-Binding Proteins; Fungal Proteins; Green Fluorescent Proteins; Luminescent Proteins; Meiosis; Microtubules; Mutation; Nuclear Proteins; Recombinant Fusion Proteins; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Spindle Apparatus; Telomere; Telomere-Binding Proteins; Transcription Factors; Ubiquitin Thiolesterase
PubMed: 11901107
DOI: 10.1093/genetics/160.3.861 -
Journal of Biomolecular NMR Apr 2012A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble...
A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS(2)) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS(2)-tag is replaced with non-isotope labeled PrS(2)-tag, silencing the NMR signals from PrS(2)-tag in isotope-filtered (1)H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS(2)-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS(2) (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS(2)-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS(2)-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone (1)H, (15)N and (13)C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear (1)H-(15)N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.
Topics: Escherichia coli; Escherichia coli Proteins; Isotope Labeling; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Protein S; Proteins; Recombinant Fusion Proteins; Ribosomal Proteins; Solubility
PubMed: 22389115
DOI: 10.1007/s10858-012-9610-0 -
Cell Jan 1995
Review
Topics: Adaptor Proteins, Signal Transducing; Calcium-Calmodulin-Dependent Protein Kinases; Carrier Proteins; Fungal Proteins; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase Kinases; Mating Factor; Models, Biological; Peptides; Pheromones; Protein Kinases; Protein Serine-Threonine Kinases; Reproduction; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces pombe Proteins; Transcription Factors; Yeasts
PubMed: 7834739
DOI: 10.1016/0092-8674(95)90402-6 -
Current Biology : CB Nov 1996The Cks proteins are essential components of the cyclin-dependent protein kinases that regulate mitosis in all eukaryotes, but their precise function remains obscure.... (Review)
Review
The Cks proteins are essential components of the cyclin-dependent protein kinases that regulate mitosis in all eukaryotes, but their precise function remains obscure. The crystal structures of several Cks proteins offer insights into their roles during the cell cycle.
Topics: Adaptor Proteins, Signal Transducing; CDC2-CDC28 Kinases; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinases; Fungal Proteins; Humans; Protein Conformation; Protein Kinases; Saccharomyces cerevisiae Proteins; Schizosaccharomyces pombe Proteins
PubMed: 8939596
DOI: 10.1016/s0960-9822(96)00741-5 -
Cell Cycle (Georgetown, Tex.) Nov 2012
Topics: Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; Meiosis; Rad51 Recombinase; Recombinases; Recombinational DNA Repair; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 23075494
DOI: 10.4161/cc.22396